Modulators of coagulation factors

ABSTRACT

The invention provides improved nucleic acid ligands that inhibit coagulation and improved modulators of the nucleic acid ligands to provide ideal modulators of coagulation. These improved nucleic acid ligands and modulators are particularly useful for inhibiting coagulation in a host undergoing a therapeutic regime such as surgery or coronary artery bypass.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/821,183 filed Jun. 22, 2007 now U.S. Pat. No. 7,723,315, allowed,which is a divisional of U.S. patent application Ser. No. 11/113,378filed Apr. 22, 2005, now U.S. Pat. No. 7,304,041, which claims thebenefit of priority to U.S. Provisional Patent Application No.60/564,873 filed Apr. 22, 2004, all of which are all incorporated hereinby reference in their entirety.

FIELD OF INVENTION

The invention is an improved agent, composition and method to regulatethe pharmacological activity of a coagulation factor with nucleic acidligands (e.g., aptamers).

BACKGROUND OF THE INVENTION

Despite substantial efforts to treat and prevent thrombotic events,arterial thrombosis continues to be the major cause of death in adultpopulations of developed nations. Although numerous medical strategiesexist for treating thrombosis, no available agent meets the therapeuticendpoints of both bioavailability and efficacy, while also having areasonable safety profile (see Feuerstein et al. (1999) Arterioscler.Thromb. Vasc. Biol. 19:2554-2562).

Under normal circumstances, an injury to vascular endothelial cellslining a blood vessel triggers a hemostatic response through a sequenceof events commonly referred to as the coagulation “cascade.” The cascadeculminates in the conversion of soluble fibrinogen to insoluble fibrinwhich, together with platelets, forms a localized clot or thrombus whichprevents extravascular release of blood components. Wound healing canthen occur followed by clot dissolution and restoration of blood vesselintegrity and flow.

Initiation of blood coagulation arises from two distinct pathways: theintrinsic and extrinsic pathways. The intrinsic pathway can be triggeredin vitro by contact of blood borne factors with artificial negativelycharged surfaces such as glass. In contrast, the extrinsic pathway canbe initiated in vivo or in vitro when tissue factor (TF), normallysequestered from the circulatory system, comes into contact with bloodafter injury. Blood exposed TF acts as a cofactor for the factor VIIa(“FVIla”) catalyzed activation of factor IX (“FIX”) and factor X (“FX”).This leads to rapid formation of FXa and thrombin, which subsequentlypolymerizes to form the fibrin clot. Both the intrinsic and extrinsicpathways are characterized by the assembly of multiple protein complexeson procoagulant surfaces, which localizes the response to the site ofinjury (see Mann, K. G. et al. (1990) Blood 76:1).

Anticoagulant Therapy

Coumarin drugs, such as warfarin as well as the glycosaminoglycans,heparin and heparan sulfate, are commonly used as anticoagulants.Warfarin, a coumarin derivative, acts by competing with vitamin Kdependent post-translational modification of prothrombin and othervitamin K-dependent clotting factors. Its action is somewhat slower andlonger lasting effect than heparin. The coumarin drugs inhibitcoagulation by inhibiting the vitamin K-dependent carboxylationreactions necessary to the function of thrombin, and factors VII, IX,and X as well as proteins C and S. These drugs act by inhibiting thereduction of the quinone derivatives of vitamin K to their activehydroquinone forms. Because of the mode of action of coumarin drugs, ittakes several days for their maximum effect to be realized. Heparinbinds to, and activates, antithrombin III which then inhibits the serineproteases of the coagulation cascade. In part due to their potency,heparin and LMW heparin suffer drawbacks. Uncontrolled bleeding is amajor complication observed in up to 7% of patients receiving continuousinfusion up to 14% of patients given intermittent bolus doses. Thetherapeutic range to achieve efficacy without placing the patient atrisk for bleeding is narrow, approximately 1 to less than 3 ugheparin/ml plasma. At concentrations greater than 4 ug/ml of heparin,clotting activity is not detectable. Thus, great care must be taken tokeep the patient's plasma concentrations within the therapeutic range.

Groups have used antibodies to coagulation factors to regulate thecoagulation cascade. For example PCT Publication No. WO 03/093422 toSchering Aktiengesellschaft discloses antibodies that bind with greateraffinity to the factor VIIa/tissue factor (FVIIa/TF) complex than totissue factor (TF) alone. These antibodies allegedly do not compete forbinding to tissue factor with Factor VII and Factor X, and inhibit FXactivation.

U.S. Pat. No. 6,001,820 to Hamilton Civic Hospitals Research DevelopmentInc. provides heparin cofactor II specific catalytic agents which arecapable of (1) selectively inactivating thrombin which is bound eitherto fibrin in a clot or to some other surface, but which has only minimalinhibitory activity against free thrombin; (2) inhibiting the assemblyof the intrinsic tenase complex and thereby the activation of Factor Xby Factor IXa; and (3) inhibiting the activation of Factor IX by FactorXIa.

Aptamers

Nucleic acids have conventionally been thought of as primarily playingan informational role in biological processes. In the past decade it hasbecome clear that the three dimensional structure of nucleic acids cangive them the capacity to interact with and regulate proteins. Suchnucleic acid ligands or “aptamers” are short DNA or RNA oligomers whichcan bind to a given ligand with high affinity and specificity. As aclass, the three dimensional structures of aptamers are sufficientlyvariable to allow aptamers to bind to and act as ligands for virtuallyany chemical compound, whether monomeric or polymeric. Aptamers haveemerged as promising new diagnostic and therapeutic compounds,particularly in cancer therapy and the regulation of blood coagulation.

Nucleic acid ligands can be identified through methods related to amethod termed the Systematic Evolution of Ligands by EXponentialenrichment (SELEX). SELEX involves selection of protein-binding nucleicacids from a mixture of candidate oligonucleotides and step-wiseiterations of binding, partitioning and amplification to achieve thedesired criterion of binding affinity and selectivity. The SELEX processwas first described by Gold and Tuerk in U.S. Pat. No. 5,475,096, andthereafter in U.S. Pat. No. 5,270,163 (see also WO 91/19813; Tuerk etal. (1990) Science 249:505-10).

A number of third parties have applied for and secured patents coveringthe identification, manufacture and use of aptamers. As stated above,Gold and Tuerk are generally credited with first developing the SELEXmethod for isolating aptamers, and their method is described in a numberof United States patents including U.S. Pat. Nos. 5,670,637, 5,696,249,5,843,653, 6,110,900, and 5,270,163. Thomas Bruice et al. reported aprocess for producing aptamers in U.S. Pat. No. 5,686,242, which differsfrom the original SELEX process reported by Tuerk and Gold because itemploys strictly random oligonucleotides during the screening sequence.The oligonucleotides screened in the '242 patent lack theoligonucleotide primers that are present in oligonucleotides screened inthe SELEX process.

Several patents to Gold et al. contain claims covering aptamers tothrombin. For example, U.S. Pat. No. 5,670,637 contains claims coveringaptamers that bind to proteins. U.S. Pat. No. 5,696,249 claims anaptamer produced by the SELEX process. U.S. Pat. Nos. 5,756,291 and5,582,981 to O'Toole, disclose and claim a method for detecting thrombinusing a labeled aptamer that comprises a defined six nucleotidesequence. U.S. Pat. Nos. 5,476,766 and 6,177,557 disclose compounds andmethods to identify nuclei acid ligand solutions to thrombin usingSELEX.

Sullenger, Rusconi, Kontos and White in WO 02/26932 describe RNAaptamers that bind to coagulation factors, E2F family transcriptionfactors, Ang1, Ang2, and fragments or peptides thereof, transcriptionfactors, autoimmune antibodies and cell surface receptors useful in themodulation of hemostasis and other biologic events. See also Rusconi etal, Thrombosis and Haemostasis 83:841-848 (2000), White et al, J. ClinInvest 106:929-34 (2000), Ishizaki et al, Nat Med 2:1386-1389 (1996),and Lee et al, Nat. Biotechnol. 15:41-45 (1997)).

Modulation of Aptamers

PCT Publication No. WO 02/096926 to Duke University describes agents andmethods to modulate the biological activity of nucleic acid ligandsthrough the administration of a modulator. The publication describesaptamers controlled by modulators that can be nucleic acids. Themodulatable aptamers are described as being useful in the treatment ofdiseases in which it is important to inhibit coagulation, elongationfactor 2 activity or angiogenesis. The modulatable aptamers to controlcoagulation include the aptamers to coagulation factors VII or VIIa,VIII or VIIIa, IX or IXa, V or Va, X or Xa, complexes formed with thesefactors, as well as platelet receptors. The modulator can change thebinding of the nucleic acid ligand for its target, degrade or otherwisecleave, metabolize or break down the nucleic acid ligand while theligand is exerting its effect. Modulators can be administered in realtime as needed based on various factors, including the progress of thepatient, as well as the physician's discretion in how to achieve optimaltherapy.

Maximizing Utility of Aptamers

In order for aptamers to be useful therapeutic reagents, they shouldbind tightly to proteins, inhibit a specified function of that proteinif an antagonist is desired and have no harmful side-effects. UnmodifiedRNA is not realistically used as a therapeutic agent since blood is richin ribonucleases. Some modification of single-stranded RNA and DNA canproduce molecules which are stable in blood and certain known aptamershave 2′F or 2′NH₂ groups within each pyrimidine nucleotide.

However, there is no way to predict how a particular modificationchanges aptamers. In particular, when additional limitations arerequired, as is the case with modulatable aptamers, no techniques existto predict how one or more modifications can affect the capacity of theaptamer to regulate its ligands and at the same time continue to beregulated by antidote binding.

The successful use of aptamers as therapeutic agents depends not only ontheir efficacy and specificity, but also on economics. Extrapolatingfrom the most successful animal experiments of currently availableaptamers, an aptamer dose of 1-2 mg/kg body wt is usually an effectivedose (derived from experiments on aptamers inhibiting VEGF, PDGF,L-selectin, and P-selectin). For a 70-kg adult, this means that eachinjected dose would be 70-140 mg. For acute indications, such as organtransplant, myocardial infarcts, toxic or septic shock, angioplasty, orpulmonary embolism treatment every 3 days for 15 days would involve$700-$1400 in cost of goods. Clearly, for chronic indications, the costof the goods is an issue. There is thus a need to reduce the cost ofmanufacturing of aptamers.

Several methods have been developed that modify the base SELEX processto obtain modified aptamers. For example, patents disclose the use ofmodified nucleotides in the SELEX process to obtain aptamers thatexhibit improved properties. U.S. Pat. No. 5,660,985 provides2′-modified nucleotides that allegedly display enhanced in vivostability. U.S. Pat. No. 6,083,696 discloses a “blended” SELEX processin which oligonucleotides covalently linked to non-nucleic acidfunctional units are screened for their capacity to bind a targetmolecule. Other patents describe post-SELEX modifications to aptamers todecrease their size, increase their stability, or increase targetbinding affinity (see, e.g., U.S. Pat. Nos. 5,817,785 and 5,648,214).

In U.S. Pat. No. 5,245,022 Weis et al. disclose an oligonucleotide ofabout 12-25 bases that is terminally substituted by apolyalkyleneglycol. These modified oligonucleotides are reported to beresistant to exonuclease activity.

U.S. Pat. Nos. 5,670,633 and 6,005,087 to Cook et al. describe thermallystable 2′-fluoro oligonucleotides that are complementary to an RNA orDNA base sequence. U.S. Pat. Nos. 6,222,025 and 5,760,202 to Cook et al.describe the synthesis of 2′-O substituted pyrimidines and oligomerscontaining the modified pyrimidines. EP 0 593 901 B1 disclosesoligonucleotide and ribozyme analogues with terminal 3′,3′- and5′,5′-nucleoside bonds. U.S. Pat. No. 6,011,020 to Gold et al. disclosesand claims an aptamer modified by polyethylene glycol.

Currently, a strong need remains to provide methods and compositions totreat patients in need of anticoagulant therapy, and in particular,during surgery or other medical intervention.

Therefore, it is an object of the present invention to provide methodsand compositions to treat patients in need of anticoagulant therapy, andin particular, during surgery or other medical intervention

It is another object of the present invention to provide more controlover the therapeutic effect, pharmacokinetics and duration of activityof anticoagulant therapies.

SUMMARY OF INVENTION

Improved nucleic acid ligands for anticoagulant therapy are disclosed aswell as improved nucleic acid ligands in combination with an antidotethat changes the binding of the nucleic acid ligand for its target orthat degrades or otherwise cleaves, metabolizes or breaks down thenucleic acid ligand while the ligand is still exerting its effect. Theseimproved aptamers provide favorable anticoagulant properties for in vivoapplications, including during human or veterinary surgery. Theanticoagulant function of the improved aptamer is convenientlyneutralized on administration of its antidote when desired by thesurgeon or other medical care specialist.

In one aspect of the invention, improved nucleic acid ligands oraptamers to a factor in the blood coagulation cascade are provided. Insome embodiments, the factors include Factor IX (FIX) or the cleavageproduct Factor IXa (FIXa). In some embodiments, the aptamers are ligandsto the complex formed by FIXa with Factor VIIIa (FVIIIa), also known asthe “intrinsic tenase complex.” In some embodiments, the aptamers areligands that inhibit the complex formation between FIXa and FVIIIa. In asubembodiment, the aptamers of the present invention bind to the complexof FIX and FVIIIa and inhibit activation of Factor X (FX). The aptamerscan interact with FIX, FIXa or a complex formed with FVIIIa in thepresence or absence of additional calcium. The aptamers can alsointeract with the factors of the complex at a cell membrane. In oneembodiment, the aptamers bind to the intrinsic tenase complex at themembrane surface.

In another aspect of the invention, the applicants have discoveredimproved aptamers to gene products of coagulation Factor IX (FIX), andto its cleavage product, Factor IXa (FIXa). In one embodiment, thenucleic acid ligand includes at least one region that binds to anotherregion in the molecule via Watson-Crick base pairing (stem) and at leastone region that does not bind to any other regions of the molecule underphysiological conditions (loop). In a further embodiment, the nucleicacid ligand includes two stems (stem 1 and stem 2) and two loops (loop 1and loop 2). Typically, stem 1 is one to fifteen or one to twentynucleotide pairs long. Stem 1 can also be ten, nine, eight, seven, six,five, four, three or two nucleotides long. In general, stem 2 is one toten nucleotides long. Stem 2 can also be nine, eight, seven, six, five,four, three or two nucleotides long. The length of loop 1 and loop 2 canalso be varied. Loop 2 can be as long as ten nucleotides, but can alsobe eight or less nucleotides long, including, seven, six, five, four,three or two nucleotides long. The length of loop 1 can also be varied,and in one embodiment includes ten or nince nucleotides in the 5′-3′direction and one nucleotide in the 3′-5′ direction.

The aptamers to a Factor IX gene product of the present invention can becomprised of ribonucleotides or deoxyribonucleotides, or a combinationthereof. In general, the improved aptamers are at least 25 nucleotideslong, and typically not longer than 35-40 nucleotides long. In oneembodiment, aptamers are at least 25, 30, 35, or 40 nucleotides inlength. In specific embodiments, the sequence of stem 1 includes 5nucleotides in the 5′-3′ direction. In a sub-embodiment, stem 1 includesthree guanine (G) residues in the 5′-3′ direction.

The improved aptamers can include a “suicide position.” In oneembodiment, this position becomes single stranded and labile uponbinding of the antidote to the improved aptamer and allows for cleavageof the improved aptamer upon binding of the antidote by enzymes in thecirculation, such as blood or liver endonucleases, thereby effectivelyeliminating the active aptamer from circulation. The suicide positioncan be at a guanine in stem 2 that is hydroxylated. In one embodiment,this nucleotide is in a double stranded configuration until bound withan antidote and becomes single stranded and available for cleavage uponbinding of the antidote.

In an embodiment, the aptamers include the nucleotide sequence gugg andthe complimentary sequence ccac. In one embodiment, the aptamer toFactor IX comprises the nucleotide sequence: gugga cuauacc gcg uaaugcugc c uccac t (SeqID 19).

Another embodiment of the invention includes an antidote oligonucleotidepaired with the aptamer of the invention. The antidote oligonucleotidecan be complementary to at least a portion of the aptamer. The antidotecan, for example, comprise the following sequences:

(5′-3′) sequence: cgcgguauaguccccau (Apt/AD; SEQ ID NO:1); (5′-3′)sequence: cgcgguauaguccc (Apt6/AD; SEQ ID NO:2); (5′-3′) sequence:cgcgguauaguccac (Apt7/AD; SEQ ID NO:3); (5′-3′) sequence:cgcgguauaguccauc (Apt8/AD; SEQ ID NO:4); (5′-3′) sequence:cgcgguauagucag (Apt9/AD; SEQ ID NO:5); (5′-3′) sequence: cgcgguauagucagg(Apt10/AD; SEQ ID NO:6); (5′-3′) sequence: cgcgguauagucagag (Apt11/AD;SEQ ID NO:7); (5′-3′) sequence: cgcgguauaguccucac (Apt14/AD; SEQ IDNO:8), or any modification or derivative thereof. In certainembodiments, the antidote consists essentially of one of the abovesequences, or consists entirely of one of the above sequences.

The antidote sequence does not need to be completely complementary tothe improved anticoagulant aptamer as long as the antidote sufficientlybinds to or hybridizes to the aptamer to neutralize its activity.

The aptamer pairs of the present invention include the followingsequences:

Aptamer Antidote augggga cuauacc gcg uaaugc ugc c uccccau tcgcgguauaguccccau (SEQ ID NO: 9) (SEQ ID NO: 1)augggga cuauaccgcguaaugcugcc uccccau t cgcgguauaguccccau (SEQ ID NO: 10)(SEQ ID NO: 1) ggga cuauaccgcguaaugcugcc uccc t cgcgguauaguccc(SEQ ID NO: 11) (SEQ ID NO: 2) gugga cuauaccgcguaaugcugcc uccac tcgcgguauaguccac (SEQ ID NO: 12) (SEQ ID NO: 3)gaugga cuauaccgcguaaugcugcc uccauc t cgcgguauaguccauc (SEQ ID NO: 13)(SEQ ID NO: 4) cuga cuauaccgcguaaugcugcc ucag t cgcgguauagucag(SEQ ID NO: 14) (SEQ ID NO: 5) ccuga cuauaccgcguaaugcugcc ucagg tcgcgguauagucagg (SEQ ID NO: 15) (SEQ ID NO: 6)cucuga cuauaccgcguaaugcugcc ucagag t cgcgguauagucagag (SEQ ID NO: 16)(SEQ ID NO: 7) gugagga cuauaccgcguaaugcugcc uccucac t cgcgguauaguccucac(SEQ ID NO: 17) (SEQ ID NO: 8)gugagga cuauacc gcg uaaugc ugc c uccucac t cgcgguauaguccucac(SEQ ID NO: 18) (SEQ ID NO: 8) gugga cuauacc gcg uaaugc ugc c uccac tcgcgguauaguccac (SEQ ID NO: 19) (SEQ ID NO: 3)

Improved aptamer-antidote pairs that are more stable and bioactive aredeveloped by including secondary modifications on either the aptamer orantidote or both. In one embodiment, the improved aptamer to Factor IXincludes one or more 2′-O-methyl modified nucleotides. In anotherembodiment, the improved aptamer contains one or more 2′-O-methyl andone or more 2′-fluoro modifications. In another embodiment, the aptamerand antidote contain no 2′-fluoro modifications. In yet anotherembodiment, the improved aptamer includes one or more 2′-O-methyl andone or more 2′-fluoro modifications on a stem. In one embodiment, atleast one guanine in stem 2 of an improved aptamer includes a hydroxylsugar (2′-OH). In another embodiment, at least one uridine in stem 1 orin stem 2 of the improved aptamer is modified with either a 2′-fluoro or2′-O-methyl. In another embodiment, at least one cytidine in stem 2 ofthe improved aptamer is 2′-fluoro modified.

The improved aptamers and antidotes can also include nucleotides thatare modified with water-soluble polymers. Such polymers can include apolyethylene glycol, polyamine, polyether, polyanhydride, polyester, orother biodegradable pharmaceutically acceptable polymer.

The invention includes the use of the improved aptamers to bind to FIX,FIXa, or the intrinsic tenase complex. This binding can be in vitro orin vivo. The result of the binding to FIX, FIXa or the tenase complexcan be to inhibit the biological activity of the proteins or complex.

In one embodiment, the improved aptamer inhibits blood coagulation bybinding to FIXa, which is derived from the same gene product as FIX. Theinvention includes administering the improved aptamers of the inventionto a mammal in need thereof to inhibit blood coagulation. Anotherembodiment of the invention provides methods of using the improvedaptamers and antidotes during a therapeutic regime.

In one embodiment, antidotes to the improved aptamers of the inventionare provided to a mammal in need thereof to reverse the anticoagulanteffects of the improved aptamers. Improved aptamers and aptamer-antidotepairs can be administered in real time as needed based on variousfactors, including the progress of the patient, as well as thephysician's discretion in how to achieve optimal therapy. Thus, thisinvention discloses an improved regulatable therapeutic regime in thecourse of nucleic acid ligand therapy for blood coagulation. In oneexample, an antidote is provide that neutralizes the effect of theimproved aptamer to turn off anticoagulant activity when desired by thephysician or other health care provider. In another embodiment, theimproved aptamers and antidotes to blood coagulation factors areadministered in sequential steps, in which the aptamers areadministered, the antidotes are used to limit the activity of theimproved aptamers, and subsequently the aptamers are re-administered toa patient in need thereof. In one embodiment, the antidote achieves thisneutralization effect by binding to or hybridizing to the improvedaptamer.

The improved aptamers can be administered to patients suffering from orat risk of suffering from a cardiovascular disease or intervention,including surgical intervention, that causes or results in acoagulation-inducing event. Examples include acute myocardial infarction(heart attack), cerebrovascular accidents (stroke), ischemia,angioplasty, CABG (coronary artery bypass grafts), cardiopulmonarybypass, thrombosis in the circuit of cardiac bypass apparatus and inpatients undergoing renal dialysis, unstable angina, pulmonary embolism,deep vein thrombosis, arterial thrombosis, and disseminatedintravascular coagulation.

The improved aptamers can also be administered to preventcoagulation-induced inflammation. It appears that early inflammation isinduced by activation of the coagulation cascade. Therefore, theimproved aptamers can be used to treat cardiovascular diseases thatinclude a inflammatory component, for example, atherosclerosis, acutecoronary syndrome (ACS), myocardial infarction which may result inreperfusion injury, or to treat adverse events associated withpost-angioplasty restenosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of proposed two dimensional configurations of AptA, 1-39 described below. FIG. 1 a is a schematic of aptamers Apt A and1-5. 1 b is a schematic of aptamers 6-11. 1 c is a schematic of aptamersApt 12-17. 1 d is a schematic of aptamers Apt 18-20, 1 e of Apt 21. 1 fis a schematic of aptamers Apt 22-29, 1 g of Apt 30-34 and 1 h of Apt35-39.

FIG. 2 is a graph of results of activated partial thromboplastin time(APTT) test assays of aptamers Apt A and Apt 1-5 (left panel) andneutralizability by antidote AptA-AD.

FIG. 3 is a graph of results of activated partial thromboplastin time(APTT) test assays of aptamers Apt 2 and 6-8 (right panel) andneutralizability by antidote (left panel).

FIG. 4 is a graph of results of activated partial thromboplastin time(APTT) test assays of aptamers Apt 2 and 9-11 (left panel) andneutralizability by antidote (right panel).

FIG. 5 is a graph of results of activated partial thromboplastin time(APTT) test assays of aptamers Apt A, 2 and 12-17 (left panel) andneutralizability by antidote (right panel).

FIG. 6 is a graph of results of activated partial thromboplastin time(APTT) test assays of aptamers Apt 2, 15, 16 and 21 (left panel) andneutralizability by antidote (right panel).

FIG. 7 is a graph of results of activated partial thromboplastin time(APTT) test assays of aptamers Apt 2 and 16-20 (left panel) andneutralizability by antidote (right panel).

FIG. 8 is a graph of results of activated partial thromboplastin time(APTT) test assays of pegylated AptA compared to pegylated Apt 16 and 19and cholesterol-modified Apt A (chol-A). Left panel is aptamer controlof coagulation factor IX and right panel is neutralizability byantidote.

FIG. 9 is a graph of results of activated partial thromboplastin time(APTT) test assays of aptamers Apt 2, 16 and 22-27 (left panel) andneutralizability by antidote (right panel).

FIG. 10 is a graph of results of activated partial thromboplastin time(APTT) test assays of aptamers Apt 2 and 30-33 (A, left panel),neutralizability by antidote of Apt 2, 30 and 33 (A, right panel), APTTtest assays of aptamers Apt 2, 30, 33 and 34 (B, left panel) andneutralizability by antidote (B, right panel).

FIG. 11 is a graph of results of activated partial thromboplastin time(APTT) test assays of aptamers Apt A, 2, 19 and 35-39 (left panel) andneutralizability by antidote of Apt A, 19, 35, 38 and 39 (right panel).

FIG. 12 is a graph of results of neutralizability by antidote assays ofaptamers Apt 2, 34, 39 and Peg-19.

FIG. 13 is a graph of the in vitro anticoagulant activity of PEG-Apt39compared to CH-AptA and PEG-AptA.

FIG. 14 are graphs of systemic anticoagulant activity (14 a) andneutralizability (14 b) of PEG-Apt39 in swine. The change in the valueof the respective clotting assays is the difference between the clottingtime at the time point and the pre-injection baseline for that animal.n=2 for PEG-Apt39 treated animals and n=3 for CH-AptA treated animals.Whole blood ACT values are shown in the bottom panel, and plasma APTTvalues in the panel at the top.

FIG. 15 is graphs of systemic anticoagulant activity (14 a) andneutralizability (14 b) of PEG-Apt39 in swine. The change in the valueof the respective clotting assays is the difference between the clottingtime at the time point and the pre-injection baseline for that animal.n=2 for PEG-Apt39 treated animals. Data from this experiment is comparedto the anticoagulation and neutralization data for PEG-Apt39 presentedin FIG. 3. Whole blood ACT values are shown in the left panel, andplasma APTT values in the panel at right.

FIG. 16 is a graph of systemic anticoagulation of monkeys by Apt39administration as described in Example 9. The level of anticoagulationin the monkeys was monitored with the APTT. For animals treated with 15mg/kg, Apt39 data are presented as the mean±SEM. For animals at the 5and 30-mg/kg dose levels, data are presented as the mean±range, as therewere only 2 animals at each of these dose levels.

FIG. 17 is a graph of the systemic anticoagulation of monkeys with Apt39and reversal with antidote Apt7AD, as described in Example 9. The levelof anticoagulation in the monkeys was monitored with the APTT. Apt7ADwas administered at t=3 hours following Apt39 administration. Data arepresented as the mean±SEM.

DETAILED DESCRIPTION Definitions

A “nucleic acid ligand” or “aptamer” is a nucleic acid that can form athree dimensional configuration, which allows it to interact as a ligandwith a target molecule. The terms refer to oligonucleotides havingspecific binding regions that are capable of forming complexes with anintended target molecule in an environment wherein other substances inthe same environment are not complexed to the oligonucleotide. Thespecificity of the binding is defined in terms of the comparativedissociation constants (K_(d)) of the aptamer for target as compared tothe dissociation constant with respect to the aptamer and othermaterials in the environment or unrelated molecules in general.Typically, the K_(d) for the aptamer with respect to the target will be10-fold, 50-fold, 100-fold, or 200-fold less than the K_(d) with respectto the unrelated material or accompanying material in the environment.

“Aptamer antidote pair” is meant to include a specified aptamer to atarget molecule, and an oligonucleotide that changes the threedimensional configuration of the aptamer so that the aptamer can nolonger interact with its target. The antidote can be an oligonucleotidecomplimentary to a portion of the aptamer. The antidote can change theconformation of the aptamer to reduce the target binding capacity of theaptamer by 10 to 100%, 20 to 100%, 25%, 40%, 50%, 60%, 70%, 80%, 90% or100%, or any percentage in the range between 10 and 100% underphysiological conditions. The antidote can also form a three dimensionalstructure with binding activity to a target molecule. This target can bethe same or different from the target of the aptamer.

“Antidote,” “Regulator” or “Modulator” refers to any pharmaceuticallyacceptable agent that can bind an aptamer and modify the interactionbetween that aptamer and modify the interaction between that aptamer andits target molecule (e.g., my modifying the structure of the aptamer) ina desired manner.

The terms “binding activity” and “binding affinity” are meant to referto the tendency of a ligand molecule to bind or not to bind to a target.The energy of said interactions are significant in “binding activity”and “binding affinity” because they define the necessary concentrationsof interacting partners, the rates at which these partners are capableof associating, and the relative concentrations of bound and freemolecules-in a solution. The specificity of the binding is defined interms of the comparative dissociation constants (K_(d)) of an antidoteof a nucleic acid ligand as compared to the dissociation constant withrespect to other materials in the environment or unrelated molecules ingeneral.

As used herein, “consensus sequence” refers to a nucleotide sequence orregion (which might or might not be made up of contiguous nucleotides)that is found in one or more regions of at least two nucleic acidsequences. A consensus sequence can be as short as three nucleotideslong. It also can be made up of one or more noncontiguous sequences,with nucleotide sequences or polymers of up to hundreds of bases longinterspersed between the consensus sequences. Consensus sequences can beidentified by sequence comparisons between individual nucleic acidspecies, which comparisons can be aided by computer programs and other,tools for modeling secondary and tertiary structure from sequenceinformation. Generally, the consensus sequence will contain at leastabout 3 to 20 nucleotides, more commonly from 6 to 10 nucleotides.

The terms “cardiovascular disease” and “cardiovascular diseases” aremeant to refer to any cardiovascular disease as would be understood byone of ordinary skill in the art. Nonlimiting examples of particularlycontemplated cardiovascular diseases include, but are not limited to,atherosclerosis, thrombophilia, embolisms, cardiac infarction (e.g.,myocardial infarction), thromboses, angina, stroke, septic shock,hypertension, hyper-cholesterolemia, restenosis and diabetes (andassociated diabetic retinopathy). Cardiovascular disease can be treatedat any stage of progression, such as treatment of early onsetcardiovascular disease as well as treatment of advanced cardiovasculardisease. A therapeutic method directed toward-inhibiting the aggravationof cardiovascular disease by modulating coagulation is also included inthe invention.

Aptamers to Factor IX

The invention provides improved nucleic acid ligands or aptamers thatregulate blood coagulation through interaction with specific factors inthe blood coagulation cascade. The invention also provides improvedaptamer-antidote pairs to regulate coagulation. The improved aptamerstarget Factor IX gene products (which include Factor IXa) and thusreduce the non-specific side effects associated with other bloodcoagulation factor targets. Most factors in the coagulation cascade arebroad spectrum proteins with a variety of physiological roles (i.e.thrombin).

The events which occur between injury and blood clot formation are acarefully regulated and linked series of reactions. In a cell-basedmodel of coagulation, initiation takes place on tissue factor-bearingcells (monocytes, macrophages, endothelial cells). In the presence ofFVIIa (complexed with tissue factor), activation of FIX and FX generatesa small amount of thrombin from prothrombin (which subsequentlyactivates FV). In the amplification phase (also referred to as thepriming phase), the small amount of thrombin generated activatesplatelets, causing release of FVa, FXIa and FVIIIa. During the finalphase of coagulation, propagation, FIXa complexes with FVIIIa,activating FX. The FXa-FVa complex, in the presence of calcium andphospholipids substrate (prothrombinase complex), leads to a “burst” ofthrombin generation.

The cell-based model of anticoagulation has been instrumental indefining coagulation protease targets. Most previous work has focused onblood coagulation through a variety of factors such as thrombin.Thrombin is a broad acting protein with effects throughout the body.Inhibitors of thrombin can therefore have unanticipated side effects inaddition to the effects on coagulation. Thrombin not only activatesendothelial cells and induces leukocyte infiltration and edema but alsoactivates astrocytes and microglia to propagate the focal inflammationand produce potential neurotoxic effects.

The inventor has determined that Factor IXa in particular represents anattractive target because of its participation in both the initiationand propagation phases of coagulation. Iteractive in vitro selectiontechniques have been used to identify oligonucleotides capable ofbinding FIXa with high affinity (K_(d) 0.65±0.2 nM). Experimentalstudies suggest that FIXa may have a critical role in thrombosis, aswell as hemostasis. Infusion of purified FIXa into rabbits inducesthrombosis (Gitel et al. (1977) PNAS 74:3028-32; Gurewich et al. (1979)Thromb. Rsch. 14:931-940). In contrast, active site-blocked FIXaprevented clot formation and reduced intra-arterial coronary thrombosis(Lowe (2001) Brit. J. Haem. 115:507-513).

Antibodies to factor IX have also been shown to interfere with thefunction of the intrinsic tenase complex, the activation of zymogenfactor IX by factor XIa and by the tissue factor:factor VIIa complex andpotently inhibit activated partial thromboplastin clotting times (APTT)in plasma of guinea pig and rat (Refino, C. J., et al, (1999) Thromb andHaemost, 82:1188-1195; Feuerstein G Z, et al. (1999) Arterioscler ThrombVasc Biol 19(10):2554-62; Toomey J R, et al. (2000) Thromb Res.100(1):73-9).

In one embodiment, the invention provides nucleic acid ligands oraptamers to a factor in the blood coagulation cascade. In someembodiments, the factors include Factor IX (FIX) or the cleavage productFactor IXa (FIXa). In some embodiments, the aptamers are ligands to thecomplex formed by FIXa with Factor VIIIa (FVIIIa), also known as the“intrinsic tenase complex.” In some embodiments, the aptamers areligands that inhibit the complex formation between FIXa and FVIIIa. In asubembodiment, the aptamers of the present invention bind to the complexof FIX and FVIIIa and inhibit activation of Factor X (FX). The aptamerscan interact with FIX, FIXa or a complex formed with FVIIIa in thepresence or absence of additional calcium. The aptamers can alsointeract with the factors of the complex at a cell membrane. In oneembodiment, the aptamers bind to the intrinsic tenase complex at themembrane surface.

In one embodiment, the applicants have discovered improved aptamers togene products of coagulation Factor IX (FIX), and to its cleavageproduct, Factor IXa (FIXa). In one embodiment, the nucleic acid ligandincludes at least one region that binds to another region in themolecule via Watson-Crick base pairing (stem) and at least one regionthat does not bind to any other regions of the molecule underphysiological conditions (loop). In a further embodiment, the nucleicacid ligand includes two stems (stem 1 and stem 2) and two loops (loop 1and loop 2). In one embodiment, stem 1 is one to twenty nucleotideslong. In a further embodiment, stem 1 is one to ten nucleotides long. Ina further sub-embodiment, stem 1 is seven, six, five, four, three or twonucleotides long. In another embodiment, stem 2 one to twentynucleotides long. In a further embodiment, stem 2 is one to tennucleotides long. In a further sub-embodiment, stem 2 is seven, six,five, four, three or two nucleotides long.

The aptamers to a Factor IX gene product of the present invention can becomprised of ribonucleotides or deoxyribonucleotides, or a combinationthereof. In general, the improved aptamers are at least 25 nucleotideslong, and typically not longer than 35-40 nucleotides long. In oneembodiment, aptamers are at least 25, 30, 35, or 40 nucleotides inlength. In specific embodiments, the sequence of stem 1 includes 5nucleotides in the 5′-3′ direction. In a sub-embodiment, stem 1 includesthree guanine (G) residues in the 5′-3′ direction.

In an embodiment, the aptamers include the consensus nucleotidesequences gugg and the complimentary sequence ccac. When a number ofindividual, distinct aptamer sequences for a single target molecule havebeen obtained and sequenced, the sequences can be examined for“consensus sequences.” As used herein, “consensus sequence” refers to anucleotide sequence or region (which might or might not be made up ofcontiguous nucleotides) that is found in one or more regions of at leasttwo aptamers, the presence of which can be correlated withaptamer-to-target-binding or with aptamer structure.

A consensus sequence can be as short as three nucleotides long. It alsocan be made up of one or more noncontiguous sequences. With nucleotidesequences or polymers of hundreds of bases long interspersed between theconsensus sequences. Consensus sequences can be identified by sequencecomparisons between individual aptamer species, which comparisons can beaided by computer programs and other, tools for modeling secondary andtertiary structure from sequence information. Generally, the consensussequence will contain at least about 3 to 20 nucleotides, more commonlyfrom 6 to 10 nucleotides. Not all oligonucleotides in a mixture can havethe same nucleotide at such position; for example, the consensussequence can contain a known ratio of particular nucleotides. Forexample, a consensus sequence might consist of a series of fourpositions wherein the first position in all members of the mixture is A,the second position is 25% A, 35% T and 40% C, the third position is Tin all oligonucleotides, and the fourth position is G in 50% of theoligonucleotides and C in 50% of the oligonucleotides.

In specific embodiments, the aptamers include the nucleotide sequencesof the following Seq ID Nos.:

SeqID Code Size Sequence  9 AptA 35mer(5′-3′) sequence: augggga cuauacc gcg uaaugc ugc c uccccau t 10 Apt135mer (5′-3′) sequence: augggga cuauaccgcguaaugcugcc uccccau t  9 Apt235mer (5′-3′) sequence: augggga cuauacc gcg uaaugc ugc c uccccau t  9Apt3 35mer (5′-3′) sequence: augggga cuauacc gcg uaaugc ugc c uccccau t 9 Apt4 35mer(5′-3′) sequence: augggga cuauacc gcg uaaugc ugc c uccccau t  9 Apt535mer (5′-3′) sequence: augggga cuauacc gcg uaaugc ugc c uccccau t 11Apt6 29mer (5′-3′) sequence: ggga cuauaccgcguaaugcugcc uccc t 12 Apt731mer (5′-3′) sequence: gugga cuauaccgcguaaugcugcc uccac t 13 Apt8 33mer(5′-3′) sequence: gaugga cuauaccgcguaaugcugcc uccauc t 14 Apt9 29mer(5′-3′) sequence: cuga cuauaccgcguaaugcugcc ucag t 15 Apt10 31mer(5′-3′) sequence: ccuga cuauaccgcguaaugcugcc ucagg t 16 Apt11 33mer(5′-3′) sequence: cucuga cuauaccgcguaaugcugcc ucagag t 10 Apt12 35mer(5′-3′) sequence: augggga cuauaccgcguaaugcugcc uccccau t 10 Apt13 35mer(5′-3′) sequence: augggga cuauaccgcguaaugcugcc uccccau t 17 Apt14 35mer(5′-3′) sequence: gugagga cuauaccgcguaaugcugcc uccucac t 17 Apt15 35mer(5′-3′) sequence: gugagga cuauaccgcguaaugcugcc uccucac t 17 Apt16 35mer(5′-3′) sequence: gugagga cuauaccgcguaaugcugcc uccucac t 17 Apt17 35mer(5′-3′) sequence: gugagga cuauaccgcguaaugcugcc uccucac t 11 Apt18 29mer(5′-3′) sequence: ggga cuauaccgcguaaugcugcc uccc t 12 Apt19 31mer(5′-3′) sequence: gugga cuauaccgcguaaugcugcc uccac t 13 Apt20 33mer(5′-3′) sequence: gaugga cuauaccgcguaaugcugcc uccauc t 18 Apt21 35mer(5′-3′) sequence: gugagga cuauacc gcg uaaugc ugc c uccucac t 18 Apt2235mer (5′-3′) sequence: gugagga cuauacc gcg uaaugc ugc c uccucac t 18Apt23 35mer (5′-3′) sequence: gugagga cuauacc gcg uaaugc ugc c uccucac t18 Apt24 35mer(5′-3′) sequence: gugagga cuauacc gcg uaaugc ugc c uccucac t 18 Apt2535mer (5′-3′) sequence: gugagga cuauacc gcg uaaugc ugc c uccucac t 27Apt26 33mer (5′-3′) sequence: gugagga cuauacc gca aucg ugc c uccucac t28 Apt27 33mer(5′-3′) sequence: gugagga cuauacc gca aucg ugc c uccucac t 29 Apt2833mer (5′-3′) sequence: gugagga cuauacc gca aucg ugc c uccucac t 30Apt29 33mer (5′-3′) sequence: gugagga cuauacc gca aucg ugc c uccucac t18 Apt30 35mer(5′-3′) sequence: gugagga cuauacc gcg uaaugc ugc c uccucac t 18 Apt3135mer (5′-3′) sequence: gugagga cuauacc gcg uaaugc ugc c uccucac t 18Apt32 35mer (5′-3′) sequence: gugagga cuauacc gcg uaaugc ugc c uccucac t18 Apt33 35mer(5′-3′) sequence: gugagga cuauacc gcg uaaugc ugc c uccucac t 18 Apt3435mer (5′-3′) sequence: gugagga cuauacc gcg uaaugc ugc c uccucac t 19Apt35 31mer (5′-3′) sequence: gugga cuauacc gcg uaaugc ugc c uccac t 19Apt36 31mer (5′-3′) sequence: gugga cuauacc gcg uaaugc ugc c uccac t 19Apt37 31mer (5′-3′) sequence: gugga cuauacc gcg uaaugc ugc c uccac t 19Apt38 31mer (5′-3′) sequence: gugga cuauacc gcg uaaugc ugc c uccac t 19Apt39 31mer (5′-3′) sequence: gugga cuauacc gcg uaaugc ugc c uccac t

In one embodiment, the aptamer to Factor IX comprises, consists, orconsists essentially of, the nucleotide sequence: gugga cuauacc gcguaaugc ugc c uccac t (SeqID 19).

Aptamer Antidotes

It is important to be able to release blood coagulation factors frominhibition. Life threatening diseases can result from over-inhibition ofthe blood coagulation factors such as Factor IX. For example, hemophiliaB results from deficiencies in factor IX. All patients with hemophilia Bhave prolonged coagulation time and decreased factor IX clottingactivity. Like hemophilia A, there are severe, moderate and mild formsof hemophilia B and reflect the factor IX activity in plasma.

Therefore, another embodiment of the invention includes an antidotepaired with the aptamer of the invention. Antidotes or modulators caninclude any pharmaceutically acceptable agent that can bind an aptamerand modify the interaction between that aptamer and its target molecule(e.g., by modifying the structure of the aptamer) in a desired manner.Examples of such antidotes include (A) oligonucleotides complementary toat least a portion of the aptamer sequence (including ribozymes orDNAzymes or peptide nucleic acids (PNAs)), (B) nucleic acid bindingpeptides, polypeptides or proteins (including nucleic acid bindingtripeptides (see, generally, Hwang et al. (1999) Proc. Natl. Acad. Sci.USA 96:12997), and (C) oligosaccharides (e.g. aminoglycosides (see,generally, Davis et al. (1993) Chapter 8, p. 185, RNA World, Cold SpringHarbor Laboratory Press, eds. Gestlaad and Atkins; Werstuck et al.(1998) Science 282:296; U.S. Pat. Nos. 5,935,776 and 5,534,408). (Seealso the following which disclose types of antidotes that can be used inaccordance with the present invention: Chase et al. (1986) Ann. Rev.Biochem. 56:103, Eichorn et al. (1968) J. Am. Chem. Soc. 90:7323, Daleet al. (1975) Biochemistry 14:2447 and Lippard et al. (1978) Acc. Chem.Res. 11:211).

In one embodiment, the antidote oligonucleotide reverses or neutralizesat least 25%, 50%, 75%, 80% or 90% of the anticoagulant activity of theaptamer. The antidote generally has the ability to substantially bind toa nucleic acid ligand in solution at antidote concentrations of lessthan one 1 μM, or less than 0.1 μM and more preferably less than 0.01μM. In one embodiment, the antidote reduces the biological activity ofthe aptamer by 50%.

Complementary Oligonucleotides

In one embodiment, the improved antidote of the invention is anoligonucleotide that comprises a sequence complementary to at least aportion of the targeted aptamer sequence. Absolute complementarity isnot required. The sequence in one embodiment has sufficientcomplementarity to be able to hybridize with the aptamer. The ability tohybridize will depend on both the degree of complementarity and thelength of the antisense nucleic acid. Advantageously, the antidoteoligonucleotide comprises a sequence complementary to 6-25 consecutivenucleotides of the targeted aptamer, preferably, 8-20 consecutivenucleotides, more preferably, 10-15 consecutive nucleotides. In specificaspects the antidote is at least 10-25 nucleotides, at least 15-25, atleast 20-25, at least 14, 17 or at least 25 nucleotides long. Theantidotes of the invention can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded.

Formation of duplexes by binding of complementary pairs of shortoligonucleotides is a fairly rapid reaction with second orderassociation rate constants generally between 1×10⁶ and 3×10⁵ M¹s¹.Stability of short duplexes can be highly dependent on the length andbase-composition of the duplex. The thermodynamic parameters forformation of short nucleic acid duplexes have been rigorously measured,resulting in nearest-neighbor rules for all possible base pairs suchthat accurate predictions of the free energy, T_(m) and thus half-lifeof a given oligoribonucleotide duplex can be calculated (e.g., Xia etal. (1998) Biochem. 37:14719; see also Eguchi et al. (1991) AntigensisRNA, Annu. Rev. Biochem. 60:631).

In a specific embodiment, the present invention provides improvedantidotes that specifically and rapidly reverse the anticoagulant andantithrombotic effects of the improved aptamers that target componentsof the coagulation pathway, in particular the aptamers of FIX and FIXa.The antidotes can be administered to reverse the aptamer activity by aphysician or other health care provider. In specific embodiments, theimproved antidotes according to the present invention are nucleic acidscorresponding to the sequences: (5′-3′) sequence: cgcgguauaguccccau(Apt/AD; SEQ ID NO:1); (5′-3′) sequence: cgcgguauaguccc (Apt6/AD SEQ IDNO:2); (5′-3′) sequence: cgcgguauaguccac (Apt7/AD; SEQ ID NO:3); (5′-3′)sequence: cgcgguauaguccauc (Apt8/AD; SEQ ID NO:4); (5′-3′) sequence:cgcgguauagucag (Apt9/AD; SEQ ID NO:5); (5′-3′) sequence: cgcgguauagucagg(Apt10/AD; SEQ ID NO:6); (5′-3′) sequence: cgcgguauagucagag (Apt11/AD;SEQ ID NO:7); (5′-3′) sequence: cgcgguauaguccucac (Apt14/AD; SEQ IDNO:8), or any modification or derivative thereof. The antidote sequencecan be at least 20%, 50%, 75% or 90% homologous to the sequence of thecorresponding aptamer. In one embodiment, the antisense sequence isseparately administered.

Antisense techniques are discussed for example, in Okano, et al. (1991)J. Neurochem. 56:560 and “Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression,” (1988) CRC Press, Boca Raton, Fla. In oneembodiment, oligonucleotide antidotes of the invention areadvantageously targeted at single-stranded regions of the aptamer. Thiscan facilitate nucleation and, therefore, the rate of aptamer activitymodulation, and also, generally leads to intermolecular duplexes thatcontain more base pairs than the targeted aptamer. The aptamer to FactorIXa of the present invention may be used to design an antisenseoligonucleotide. The antisense oligonucleotide hybridizes to the aptamerin vivo and blocks the binding of the aptamer to factor IXa.

To design an improved antidote to the improved aptamers of theinvention, various strategies can be used to determine the optimalbinding site. The complimentary oligonucleotides can be “walked” aroundthe aptamer. This “walking” procedure includes, after a minimalconsensus ligand sequence has been determined for a given improvedaptamer, adding random sequence to the minimal consensus ligand sequenceand evolving additional contacts with the target, such as in separatebut adjacent domains. A walking experiment can involve two experimentsperformed sequentially. A new candidate mixture is produced in whicheach of the members of the candidate mixture has a fixed nucleicacid-region that corresponds to a nucleic acid ligand of interest. Eachmember of the candidate mixture also contains a randomized region ofsequences. According to this method it is possible to identify what arereferred to as “extended” nucleic acid ligands, which contain regionsthat can bind to more than one binding domain of a target.

Changes in the sugar can affect antidote stability, in part becausesugar modifications that result in RNA-like oligonucleotides, e.g.,20-fluoro or 20-methoxy, do not appear to serve as substrates for RNaseH. Alterations in the orientation of the sugar to the base can alsoaffect RNase H activation. Additionally, backbone modificationsinfluence the ability of oligonucleotides to activate RNase H.Methylphosphonates do not activate it, whereas phosphorothioates areexcellent substrates. In addition, chimeric molecules have been studiedas oligonucleotides that bind to RNA and activate RNase H. For example,oligonucleotides comprising wings of 20-methoxy phosphonates and afive-base gap of deoxyoligonucleotides bind to their target RNA andactivate RNase H.

In one embodiment, 2′-O-methyl modified antidotes (e.g., 2′-O-methyloligonucleotides) about 15 nucleotides in length can be used, thecomplementarity of which is staggered by about 5 nucleotides on theaptamer (e.g., oligonucleotides complementary to nucleotides 1-15, 6-20,11-25, etc.). The impact of tertiary structure of the improved aptameron the efficiency of hybridization is difficult to predict. Assaysdescribed in the Examples that follow can be used to assess the abilityof the different oligonucleotides to hybridize to a specific aptamer.The ability of the different oligonucleotide antidotes to increase therate of dissociation of the aptamer from, or association of the aptamerwith, its target molecule can also be determined by conducting kineticstudies using, for example, BIACORE assays. Oligonucleotide antidotescan be selected such that a 5-50 fold molar excess of oligonucleotide,or less, is required to modify the interaction between the aptamer andits target molecule in the desired manner.

The antidotes of the invention may be conjugated to another molecule,e.g., a peptide, hybridization triggered cross-linking agent, transportagent, hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may optionally comprise at least onemodified base moiety, including but not limited to one selected from5-fluorouracil, 5-fluorocytosine, 5-bromouracil, 5-bromocytosine,5-chlorouracil, 5-chlorocytosine, 5-iodouracil, 5-iodocytosine,5-methylcytosine, 5-methyluracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylamin-O-methyl thiouridine,5-carboxymethylamin-O-methyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 6-methylcytosine, N6-adenine,7-methylguanine, 5-methylamin-O-methyluracil,5-methoxyamin-O-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil, 5-methoxycytosine,2-methylthio-N&isopentenyladenine, uracil oxyacetic acid (v),butoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil oxyacetic acid (v), 5-methyl thiouracil,3-(3-amino-3-N carboxypropyl) urdeil, (acp3)w, and 2,6-diaminopurine.

The antidotes may also include at least one modified sugar moietyselected from the group including, but not limited to, arabinose,2-fluoroarabinose, xylulose, hexose, 2′-fluororibose, 2′-O-methylribose,2′-O-methoxyethylribose, 2′-O-propylribose, 2′-O-methylthioethylribose,2′-O-diethylaminooxyethylribose, 2′-O-(3-aminopropyl)ribose,2′-O-(dimethylaminopropyl)ribose, 2′-O-(methylacetamido)ribose, and2′-O-(dimethylaminoethyloxyethyl)ribose. In yet another embodiment, theantisense oligonucleotide comprises at least one modified phosphatebackbone selected from the group including, but not limited to, aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiainidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

Ribozymes and DNAzymes

Improved aptamers or antidotes can also be enzymatic nucleic acids. Sucha ribozyme or DNAzyme act by first binding to a target RNA or DNA (seeCech U.S. Pat. No. 5,180,818) and then cleaving the target. An enzymaticnucleic acid can repeatedly bind and cleave new targets thereby allowingfor inactivation of RNA aptamers. There are at least five classes ofribozymes that each display a different type of specificity. In the caseof antidotes, this enzymatic activity can complement or substitute forthe introduction of a “suicide position” in the improved aptamer.

The enzymatic nature of a ribozyme may be advantageous over othertechnologies because the effective concentration of ribozyme necessaryto effect a therapeutic treatment is lower than that of an antisenseoligonucleotide. A single ribozyme molecule is able to cleave manymolecules of target RNA. In addition, the ribozyme is a highly specificinhibitor, with the specificity of inhibition depending not only on thebase pairing mechanism of binding, but also on the mechanism by whichthe molecule inhibits the expression of the RNA to which it binds. Thatis, the inhibition is caused by cleavage of the RNA target and sospecificity is defined as the ratio of the rate of cleavage of thetargeted RNA over the rate of cleavage of non-targeted RNA. Thiscleavage mechanism is dependent upon factors additional to thoseinvolved in base pairing. Thus, it may be that the specificity of actionof a ribozyme is greater than that of antisense oligonucleotide bindingthe same RNA site.

Another class of catalytic molecules are called “DNAzymes”. DNAzymes aresingle-stranded, and cleave both RNA and DNA. A general model for theDNAzyme has been proposed, and is known as the “10-23” model. DNAzymesfollowing the “10-23” model, also referred to simply as “10-23DNAzymes”, have a catalytic domain of 15 deoxyribonucleotides, flankedby two substrate-recognition domains of seven to ninedeoxyribonucleotides each. In vitro analyses show that this type ofDNAzyme can effectively cleave its substrate RNA at purine:pyrimidinejunctions under physiological conditions. As used herein, “DNAzyme”means a DNA molecule that specifically recognizes and cleaves a distincttarget nucleic acid sequence, which may be either DNA or RNA.

Adapted Nucleic Acids

In another aspect of the invention, the antidote to the improvedaptamers are Peptide Nucleic Acids (PNAs). PNAs are compounds that areanalogous to oligonucleotides, but differ in composition in that thedeoxyribose backbone of oligonucleotide is replaced by a peptidebackbone. Each subunit of the peptide backbone is attached to anaturally-occurring or non-naturally-occurring nucleobase.

PNAs can be advantageous as fast acting antidotes because they bind moretightly to the corresponding improved aptamers than theirnon-substituted oligonucleotide counterparts. PNAs bind to both DNA andRNA and the resulting PNA/DNA or PNA/RNA duplexes are bound tighter thancorresponding DNA/DNA or DNA/RNA duplexes as evidenced by their highermelting temperatures (T_(m)). Another advantage of PNA/DNA(RNA) duplexesis that T_(m) is practically independent of salt concentration. SincePNAs are an analogue of DNA in which the backbone is a pseudopeptiderather than a sugar, they mimic the behaviour of DNA and bindscomplementary nucleic acid strands.

PNAs are synthetic polyamides comprised of repeating units of the aminoacid, N-(2-aminoethyl)-glycine, to which the nucleobases adenine,cytosine, guanine, thymine and uracil are attached through a methylenecarbonyl group. Natural and unnatural nucleobases, such as pseudoisocytosine, 5-methyl cytosine and 2,6-diaminopurine, inosine, uracil,5-methylcytosine, thiouracil, 2,6-diaminopurine, bromothymine,azaadenines or azaguanines among many others, also can be incorporatedin PNA synthons. PNAs are most commonly synthesized from monomers (PNAsynthons) protected according to the t-Boc/benzyl protection strategy,wherein the backbone amino group of the growing polymer is protectedwith the t-butyloxycarbonyl (t-Boc) group and the exocyclic amino groupsof the nucleobases, if present, are protected with the benzyloxycarbonyl(benzyl) group. PNA synthons protected using the t-Boc/benzyl strategyare now commercially available.

Morpholino nucleic acids (MNAs) can also be advantageous in antidotepreparation because morpholinos are completely resistant to nucleasesand they appear to be free of most or all of the non-antisense effectsthat plague S-DNAs. MNAs are assembled from morpholino subunits, each ofwhich contains one of the four genetic bases (adenine, cytosine,guanine, and thymine) linked to a 6-membered morpholine ring. Subunitsof are joined by non-ionic phosphorodiamidate intersubunit linkages togive a MNA. These MNAs can have substantially better antisenseproperties than do RNA, DNA, and their analogs having 5-membered riboseor deoxyribose backbone moieties joined by ionic linkages (seewwwgene-tools.com/Morpholinos/body_morpholinos.HTML).

U.S. Pat. No. 6,153,737 to Manoharan et al. is directed to derivatizedoligonucleotides wherein the linked nucleosides are functionalized withpeptides, proteins, water soluble vitamins or lipid soluble vitamins.This disclosure was directed towards antisense therapeutics bymodification of oligonucleotides with a peptide or protein sequence thataids in the selective entry of the complex into the nuclear envelope.Similarly, water-soluble and lipid-soluble vitamins can be used toassist in the transfer of the anti-sense therapeutic or diagnostic agentacross cellular membranes.

Locked nucleic acids (LNAs) can also be used to preparet the antidotesof the present invention. LNAs are a novel class of DNA analogues thatpossess certain features that make them prime candidates for improvingnucleic acid properties. The LNA monomers are bicyclic compoundsstructurally similar to RNA-monomers. LNAs share most of the chemicalproperties of DNA and RNA, are water-soluble, can be separated by gelelectrophoreses, ethanol precipitated, etc. (Tetrahedron, 54, 3607-3630(1998)). However, introduction of LNA monomers into either DNA or RNAoligos results in high thermal stability of duplexes with complementaryDNA or RNA, while, at the same time obeying the Watson-Crickbase-pairing rules. This high thermal stability of the duplexes formedwith LNA oligomers together with the finding that primers containing 3′located LNA(s) are substrates for enzymatic extensions, e.g. the PCRreaction, makes these compounds suitable for the antidotes of thepresent invention. For examples of LNAs see U.S. Pat. No. 6,316,198.

In other embodiments, the stabilized nucleic acid can be a PCO(pseudocyclic oligonucleobase), or a 2′-O,4′-C-ethylene bridged nucleicacid (ENA).

Modifications

The improved aptamers and aptamer-antidote combinations of the presentinvention are modified by substituting particular sugar residues, bychanging the composition of the aptamer and the size of particularregions in the aptamer, and by designing aptamers that can be moreeffectively inhibited by antidotes. The design of aptamers includes anappreciation for the secondary structure of the aptamer (see FIG. 1) andthe relationship between the secondary structure and the antidotecontrol. Unlike conventional methods of modifying nucleic acids, thedesign of the improved aptamers to FIX gene products included in theinvention must include a consideration of the antidote control.Controlled aptamers require that the aptamer be stable in circulationbut not so stable that it is not antidote controlled. The aptamers canbe modified by truncation, but antidotes need to be designed to controleach aptamer when truncated. Further, certain modifications,particularly at the interface of the stems and loops cannot be modifiedfrom 2′-fluoro or the aptamer can lose activity.

In one embodiment, the design includes decreasing the 2′-hydroxylcontent of the aptamer or the antidote, or both. In another embodiment,the design includes decreasing the fluoro content of the aptamer or theantidote, or both. In a further embodiment, the design includesincreasing the O-methyl content of the aptamer or the antidote, or both.In a further embodiment, the design includes decreasing the size of theaptamer. In another embodiment, the size of the antidote is changed inrelation to the size of the aptamer. In yet another embodiment, guaninestrings are reduced to less than four guanine, or less than threeguanine, or less than two guanine or no guanines. However, the jointeffect of these changes must meet the challenge of creating ananticoagulant that provides adequate activity but is easily neutralizedby the antidote.

Yet another embodiment includes a method of designing aptamers with a“suicide position” which allows more effective regulation by pairedantidotes. In one embodiment, this position becomes single stranded andlabile upon binding of the antidote to the improved aptamer and allowsfor cleavage of the improved aptamer upon binding of the antidote byenzymes in the circulation, such as blood or liver endonucleases,thereby effectively eliminating the active aptamer from circulation. Thesuicide position can be, in one embodiment, at a guanine in stem 2 thatis hydroxylated. In one embodiment, the aptamer is in a double strandedconfiguration until bound with an antidote and becomes single strandedand available for cleavage upon binding of the antidote.

The applicants have discovered aptamer-antidote pairs that are stableand bioactive by including secondary modifications on either the aptameror antidote or both. In specific embodiments, the aptamers to Factor IXinclude modified nucleotides. In one embodiment, the aptamer containsone or more 2′-O-methyl groups. In another embodiment, the aptamer andantidote contain one or more 2′-O-methyl and one or more 2′-fluoromodifications. In another embodiment, the aptamer and antidote containno 2′-fluoro modifications. In yet another embodiment, the aptamerincludes one or more 2′-O-methyl and one or more 2′-fluoro modificationson its stem. The aptamers can also include nucleotides that are modifiedwith soluble polymers. Such polymers can include polyethylene glycol,polyamines, polyesters, polyanhydrides, polyethers or other watersoluble pharmaceutically acceptable polymer.

Purines within given aptamer sequence of FIX inhibitor can toleratesubstitution of 2′-O-methyl sugars for current 2′ hydroxyl sugars(Example 1, FIG. 1). Applicants found that the aptamers fall into threeclasses: (1) gain of anticoagulant activity (Apt-4); (2) moderate lossof activity (Apt-1, 2, and 3); and (3) severe loss of activity (Apt 5)(FIG. 2). Data from Apt-5 indicates that the impact of whollysubstituting 2′-O-methyl purines for 2′ hydroxyl purines issignificantly greater than any individual sector substitution alone(FIG. 2). In the case of this aptamer, it is possible that this suggestspotential interaction between sectors, or that impairment caused bysubstitution within one of the sectors is exacerbated by additionalmodifications (ie. one of the sectors is an Achilles heel). The enhancedantidote control exhibited by Apt-1, 2 and 3 suggests that introductionof 2′O-methyl residues within the antidote binding site improves theability of the antidote oligonucleotide to bind to the aptamer. This isconsistent with the increase in thermodynamic stability observed forduplexes containing 2′-O-methyl RNA residues in each strand, andsuggests that duplexes of 2′-O-methyl-2′-O-methyl strands are morethermodynamically stable than duplexes composed of 2′O-methyl-2′ fluorostrands. An alternative conclusion is that the reduction in activity ofApt-1, 2 and 3 leads to more “free” aptamer in the plasma at any giventime, which is thus more readily bound by the antidote oligonucleotide

In another embodiment, aptamers of the present invention can includemodified pyrimidine nucleosides. Replacing 2′ fluoropyrimidines with2′-O-methyls within stem 1 improved activity and yielded a compound thattolerates a greater level of substitution. Comparison of the activity ofApt 30 and 33 to Apt 31 and 32 demonstrates that C16 needs to contain a2′ fluoro sugar and G25 a 2′ hydroxyl sugar (FIG. 16 a). Activityobserved between Apt 31 and 32 suggests that remaining positions withinstem 2 can contain 2′-O-methyl sugars. In fact, Apt 31 appears topossess slightly greater potency than Apt 32, indicating that a compoundwith 2′ fluoro at C16, 2′hydroxyl at G25, and the remaining residues2′-O-methyl may exhibit greater potency than Apt 33. Apt 33 is morereadily neutralizable than Apt 30, suggesting additional 2′-O-methylresidue within the antidote-binding site of the aptamer improvesantidote binding. Apt 34 have C16 as a 2′ fluoro rather than 2′-O-methylnucleoside (FIG. 16 b). Substitution increased anticoagulant activity(compare Apt 34 to Apt 33) but did result in a modest loss of“neutralizability”, although 34 still requires a lower excess ofantidote to achieve 90% neutralization (˜5:1 vs 10:1) than the parentalAptA compound (FIG. 16 b). Both results are consistent with an increasein the stability of stem 2 due to 2′-O-methyl substitution. It issurprising that others have not obviously pursued 2′-O-methylsubstitution of 2′-fluoropyrimidines, as such substitution reduces costof synthesis and appears to enable increased aptamer modification due toincreased stem stability.

In one embodiment, at least on guanine in stem 2 of an aptamer includesa hydroxyl sugar (2′-OH). In one embodiment, at least one uridine instem 1 or stem 2 is a modified base. This can be either a 2′-fluoro(2′-F) or 2′-O-methyl (2′-OCH₃) modification. In one embodiment, atleast one uridine in stem 1 or stem 2 is 2′-O-methyl modified. In oneembodiment, at least one cytidine in stem 2 is modified. In oneembodiment, at least on cytidine in stem 2 is 2′-Fluoro modified.

However, comparison of the anticoagulant activity of Apt 12 with Apt 13and 17 (FIG. 6) demonstrates that the loss of activity observed for Apt6-11 is due to the presence of 2′-O-methyl substitutions at one or morecritical residues (FIG. 7). Comparison of the anticoagulant activity ofApt 14 to Apt 12 indicates that the stretch of 4 consecutive guanosineswithin stem 1 can be altered without a significant impact onanticoagulant activity. Comparison of Apt 15 and 16 with Apt 2, 12 and17 a) demonstrates that the presence of 2′-O-methyl sugars at eachposition within stem 1 except for the closing A-U pair at the top ofstem 1 enhances activity; and b) demonstrates that the sugar of the U inthis base pair must be 2′-fluoro for the aptamer to retain potency; andc) suggests that the sugar of the A in this base pair can be a2′-O-methyl sugar without a significant impact on anticoagulantactivity. In fact, Apt 16 retains essentially full potency.

Data suggests that the antidote can more readily bind the aptamer whenstem 1 is a 2′-O-methyl-2′ fluoro stem as opposed to when both strandsof the duplex contain largely 2′-O-methyl residues. This is againconsistent with the notion that duplexes composed of 2′-O-methylresidues in both strands are more stable than those composed of alargely 2′-O-methyl strand and a largely 2′ fluoro strand. The enhancedanticoagulant activity of Apt 16 vs. Apt 15 is also consistent withthis. Alternatively, the difference in neutralizability between 14, 15and 16 could be due to the enhanced potency of Apt 16 compared to thesetwo compounds. Regardless, all are neutralized at least as well as AptA.Based upon the observation that the anticoagulant activity of Apt 14 and15 were similar, the sugar of the A at the stop of stem 1 was2′-O-methyl substituted (Apt 21, FIG. 8).

Substitution of a 2′-O-methyl sugar at this adenosine residue is welltolerated in the background of a largely 2′-O-methyl stem (FIG. 9). Infact, the potency of Apt 21 is intermediate between Apt 16 and 15.Antidote neutralization of Apt 21 is enhanced as compared to Apt 16 (seeespecially the 2.5:1 and 5:1 AD:Drug data points in FIG. 9).

Sugar modifications may ensure stability but they do not guaranteeadequate pharmacokinetics for aptamers to be therapeutically active. Inhealthy individuals, aptamers are cleared from plasma within minutes ofIV injection, probably through renal excretion. Keeping intact aptamersin the blood from hours to days after injection has been accomplished byconjugating them to larger macromolecules such as polyethyleneglycol(PEG). In another embodiment, aptamer plasma clearance has also beendecreased by embedding them in liposomes.

Nucleic acid aptamers of the present invention can also be modified byvarying the stem and loop sizes. Two families of aptamers with four,five, or six 2-O-methyl modified base pair stem 1 regions showed varyinglevels of anticoagulant activity and antidote control (see Example 2,FIGS. 3-5). Stem 1 mutants (FIG. 3) exhibit a loss of anticoagulantactivity as measured in the APTT assay (FIGS. 4 and 5). All stem 1variants exhibit less activity than the fully 2′-O-methyl purine/2′fluoro pyrimidine compound Apt 5, suggesting that one of the pyrimidineswithin stem 1 must contain a 2′ fluoro sugar for the compound to retainpotency. However, all exhibit similar activity levels suggesting stemlength may not cause loss of activity. However, 5 base pair stem 1constructs (Apt 10 and 7) do appear to be more readily antidotecontrolled than six base pair. Data suggests that a stem 1 of 5 basepairs may be preferable to those composed of 4, 6 or 7 base pairs toenhance antidote neutralization.

For targeting of an antidote, an improved aptamer can also be modifiedso as to include a single-stranded tail (3′ or 5′) in order to promoteassociation with an oligonucleotide antidote. Suitable tails cancomprise 1 to 20 nucleotides, preferably, 1-10 nucleotides, morepreferably, 1-5 nucleotides and, most preferably, 3-5 nucleotides (e.g.,modified nucleotides such as 2′-O-methyl sequences). Tailed aptamers canbe tested in binding and bioassays (e.g., as described below) to verifythat addition of the single-stranded tail does not disrupt the activestructure of the aptamer. A series of oligonucleotides (for example,2′-O-methyl oligonucleotides) that can form, for example, 1, 3 or 5basepairs with the tail sequence can be designed and tested for theirability to associate with the tailed aptamer alone, as well as theirability to increase the rate of dissociation of the aptamer from, orassociation of the aptamer with, its target molecule. Scrambled sequencecontrols can be employed to verify that the effects are due to duplexformation and not non-specific effects.

The oligonucleotide antidotes can be administered directly (e.g., aloneor in a liposomal formulation or complexed to a carrier, e.g. PEG)) (seefor example, U.S. Pat. No. 6,147,204, U.S. Pat. No. 6,011,020).Surprisingly, the addition of a PEG molecule does not reduce aptamerbinding to Factor IX the shorter the length of stem 1, and, in fact, ashorted stem 1 with pegylation do appears to increase neutralizability,providing a potentially more effective therapeutic. FIG. 10 shows theactivity and neutralizability of a pegylated aptamer with a 5 base pairstem (Apt 19). Apt 19 possesses anticoagulant activity very similar topegylated Apt16 with a 7 base pair stem 1, but ˜90% of its activity canbe neutralized with only a 2.5:1 excess of antidote to drug.

Therefore, in one embodiment, the improved aptamer or antidotes can beattached to a non-immunogenic, high molecular weight compound such aspolyethylene glycol (PEG) or other water soluble pharmaceuticallyacceptable polymer as described herein. In one embodiment, the aptameror antidote is associated with the PEG molecule through covalent bonds.Where covalent attachment is employed, PEG may be covalently bound to avariety of positions on the improved aptamer or antidote. In anotherembodiment, an oligonucleotide aptamer or antidote is bonded to the5′-thiol through a maleimide or vinyl sulfone functionality. In oneembodiment, a plurality of improved aptamers or antidotes can beassociated with a single PEG molecule. The improved aptamers andantidotes can be the same or different sequences and modifications. Inyet a further embodiment, a plurality of PEG molecules can be attachedto each other. In this embodiment, one or more aptamers or antidotes tothe same target or different targets can be associated with each PEGmolecule. In embodiments where multiple aptamers or antidotes specificfor the same target are attached to PEG, there is the possibility ofbringing the same targets in close proximity to each other in order togenerate specific interactions between the same targets. Where multipleaptamers or antidotes for specific for different targets are attached toPEG, there is the possibility of bringing the distinct targets in closeproximity to each other in order to generate specific interactionsbetween the targets. In addition, in embodiments where there areaptamers or antidotes to the same target or different targets associatedwith PEG, a drug can also be associated with PEG. Thus the complex wouldprovide targeted delivery of the drug, with PEG serving as a Linker.

The aptamers or antidotes of the invention can also include otherconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups of the invention includepolyamines, polyamides, polyethylene glycols, polyethers, groups thatenhance the pharmacodynamic properties of oligomers, and groups thatenhance the pharmacokinetic properties of oligomers. Groups that enhancethe pharmacodynamic properties, in the context of this invention,include groups that improve oligomer bioavailability, enhance oligomerresistance to degradation, and/or strengthen sequence-specifichybridization with RNA.

In specific embodiments, the aptamers include the nucleotide sequencesof any of the following sequences. (“A” is 2′OH A; “a” is 2′-O-methyl A;“G” is 2′-OH G; “g” is 2′-O-methyl G; “C” is 2′-Fluoro C; “c” is2′-O-methyl C; “U” is 2′Fluoro U; “u” is 2′-O-methyl U; and “T” isinverted 2′H T.)

SeqID Code Sequence 20 AptA AUGGGGA CUAUACC GCG UAAUGC UGC C UCCCCAU T21 Apt1 aUgggga CUAUACCGCGUAAUGCUGCC UCCCCaU T 22 Apt2AUGGGGA CUaUaCC GCG UAAUGC UGC C UCCCCAU T 23 Apt3AUGGGGA CUAUACC gCg UAAUGC UgC C UCCCCAU T 24 Apt4AUGGGGA CUAUACC GCG UaaUgC UGC C UCCCCAU T 25 Apt5aUgggga CUaUaCC gCg UaaUgC UgC C UCCCCaU T 26 Apt6ggga CUaUaCCGCGUAAUGCUGCC uccc T 27 Apt7gugga CUaUaCCGCGUAAUGCUGCC uccac T 28 Apt8gaugga CUaUaCCGCGUAAUGCUGCC uccauc T 29 Apt9cuga CUaUaCCGCGUAAUGCUGCC ucag T 30 Apt10ccuga CUaUaCCGCGUAAUGCUGCC ucagg T 31 Apt11cucuga CUaUaCCGCGUAAUGCUGCC ucagag T 32 Apt12aUgggga CUaUaCCGCGUAAUGCUGCC UCCCCaU T 33 Apt13augggga CUaUaCCGCGUAAUGCUGCC uccccau T 34 Apt14gUgagga CUaUaCCGCGUAAUGCUGCC UCCUCaC T 35 Apt15gUgaggA CUaUaCCGCGUAAUGCUGCC UCCUCaC T 36 Apt16gugaggA CUaUaCCGCGUAAUGCUGCC Uccucac T 37 Apt17gugagga CUaUaCCGCGUAAUGCUGCC uccucac T 38 Apt18gggA CUaUaCCGCGUAAUGCUGCC Uccc T 39 Apt19guggA CUaUaCCGCGUAAUGCUGCC Uccac T 40 Apt20gauggA CUaUaCCGCGUAAUGCUGCC Uccauc T 41 Apt21gugagga CUaUaCC GCG UAAUGC UGC C Uccucac T 42 Apt22gugaggA CUaUaCC gCg UAAUGC UgC C Uccucac T 43 Apt23gugaggA CUaUaCC GCG UaaUgC UGC C Uccucac T 44 Apt24gugaggA CUaUaCC gCg UaaUgC UgC C Uccucac T 45 Apt25gugaggA CUaUaCC GCg UaaUgC UgC C Uccucac T 46 Apt26gugaggA CUaUaCC gCa AUCG UgC C Uccucac T 47 Apt27gugaggA CUaUaCC GCA aUCg UGC C Uccucac T 48 Apt28gugaggA CUaUaCC gCa aUCg UgC C Uccucac T 49 Apt29gugaggA CUaUaCC GCa aUCg UgC C Uccucac T 50 Apt30CUaUaCC gCG UaaUgC UGC C Uccucac T 51 Apt31gugagga CUaUaCC gcg UaaUgC ugc C Uccucac T 52 Apt32gugagga CUaUaCC gcg UaaUgC UgC C Uccucac T 53 Apt33gugagga CUaUaCC gCg UaaUgC UGC C Uccucac T 54 Apt34gugagga CUaUaCC gCg UaaUgC uGc C Uccucac T 55 Apt35gugga CUaUaCC gCG UaaUgC UGC C Uccac T 56 Apt36gugga CUaUaCC gCG UaaUgC ugc C Uccac T 57 Apt37gugga CUaUaCC gCG UaaUgC UgC C Uccac T 58 Apt38gugga CUaUaCC gCg UaaUgC UGC C Uccac T 59 Apt39gugga CUaUaCC gCg UaaUgC uGc C Uccac T

In one specific embodiment, the aptamer to Factor IX comprises,consists, or consists essentially of, the nucleotide sequence: guggaCUaUaCC gCg UaaUgC uGc C Uccac T (Apt39; SEQ ID NO: 59).

The improved aptamers described herein can be manufactured usingtechniques known in the art. For example, U.S. patents have issued thatdescribe methods of large scale manufacturing that can be used tomanufacture aptamers. Caruthers et al., for example, describe in U.S.Pat. Nos. 4,973,679; 4,668,777; and 4,415,732 a class of phosphoramiditecompounds that are useful in the manufacture of oligonucleotides. Inanother series of patents, Caruthers et al. disclose a method ofsynthesizing oligonucleotides using an inorganic polymer support. See,e.g., U.S. Pat. Nos. 4,500,707, 4,458,066 and 5,153,319. In stillanother series of patents, Caruthers et al. discloses a class ofnucleoside phosphorodithioates that can be used to manufactureoligonucleotides. See, e.g., U.S. Pat. Nos. 5,278,302, 5,453,496 and5,602,244.

Methods of Use

Regulating Coagulation with an Improved Aptamer

The invention includes the use of improved aptamers to bind to FIX,FIXa, or the intrinsic tenase complex. The binding can be in vitro or invivo. The result of the binding to FIX, FIXa or the tenase complex canbe to inhibit the biological activity of the proteins or complex. Theimproved aptamers can be used to treat diseases such as deep venousthrombosis, arterial thrombosis, post surgical thrombosis, coronaryartery bypass graft (CABG), percutaneous transdermal coronaryangioplasty (PTCA), stroke, tumour metastasis, inflammation, septicchock, hypotension, ARDS, pulmonary embolism, disseminated intravascularcoagulation (DIC), vascular restenosis, platelet deposition, myocardialinfarction, angiogenesis, or the prophylactic treatment of mammals withatherosclerotic vessels at risk for thrombosis.

In one embodiment, the improved aptamer inhibits blood coagulation bybinding to FIXa. The invention includes administering the aptamer of theinvention to a mammal, for example, a human, in need thereof to inhibitblood coagulation. Another embodiment of the invention provides methodsof using aptamers that are well suited for administration during atherapeutic regime.

A method of improved regulating coagulation in a mammal in need thereofis provided. In one embodiment, the method comprises: (a) administeringto a warm-blooded vertebrate or mammal in need thereof, an effectiveamount of an improved aptamer that selectively binds coagulation pathwayFIX, FIXa, or the intrinsic tenase complex, or inhibits a subunit of theintrinsic tenase complex (i.e. FIX, FIXa, FVIII binding to or activationof FX); (b) modulating the biological activity of the coagulationpathway factor in the warm-blooded vertebrate through the administeringof the aptamer in step (a); and (c) providing an improved antidote toreverse the effects of the aptamer. In certain embodiments, thewarm-blooded vertebrate or mammal is a human.

As used herein, the term “mammal” is meant to include any human ornon-human mammal, including but not limited to porcine, ovine, bovine,rodents, ungulates, pigs, sheep, lambs, goats, cattle, deer, mules,horses, monkeys, dogs, cats, rats, and mice.

An important area for consideration is plasma half-life. Modificationscan alter the half-life in vivo of the improved aptamers from a fewminutes to 12 or more hours. The improved aptamers of the presentinvention can be used to treat percutaneous coronary interventions wherevascular injury occurs at a specific time and place, yielding a suddenbut relative brief prothrombotic stimulus. This may also be the casefollowing carotid angioplasty. In another embodiment, the improvedaptamers can be used in extracorporeal circulation utilized in coronarybypass grafting and hemodialysis. The latter condition is somewhatcomplex because of the inherent thrombogenicity of arteriovenous (AV)shunts. The aptamers of the invention also can be used to treat a venousthromboembolic disease, mechanical heart valve replacement, atrialfibrillation, and conceivably in either primary or secondary preventionof cardiovascular events among patients with prior events, anunfavorable risk profile, documented multibed vascular disease, vascularinflammation (early stages of atherosclerotic vasculopathy).

A method of treating cardiovascular disease in a warm-blooded vertebrateis also provided. The method comprises administering an effective amountof an improved aptamer to a vertebrate subject suffering fromcardiovascular disease that selectively binds a coagulation pathwayfactor IX, IXa, or the intrinsic tenase complex, or inhibits a subunitof the intrinsic tenase complex (i.e. FIX, FIXa, FVIII binding to oractivation of FX). Administration of the aptamer treats thecardiovascular disease in the vertebrate subject. The method can furthercomprise providing an antidote to reverse the effects of the improvedaptamer by administration of an antidote.

The improved aptamers can be administered to mammals who require bloodcoagulation therapy. The invention provides methods of treating mammalswith an aptamer to inhibit blood coagulation. The paired antidote can beadministered to reverse the effects of the aptamer. A benefit of thisdiscovery is that blood coagulation can be controlled in real time anddoes not rely on the mammal's own metabolism.

The compositions and methods of the present invention are particularlyuseful for preventing thrombosis in the circuit of cardiac bypassapparatus and in patients undergoing renal dialysis, and for treatingpatients suffering from or at risk of suffering from thrombus-relatedcardiovascular conditions, such as unstable angina, acute myocardialinfarction (heart attack), cerebrovascular accidents (stroke), pulmonaryembolism, deep vein thrombosis, arterial thrombosis, CABG surgery anddisseminated intravascular coagulation.

In addition, the improved aptamers and antidotes of the presentinvention can inhibit other cardiovascular disease associated with FIXor FIX-regulated cascades. Coagulation plays an important part inischaemic cardiovascular disease. Results of studies have shown thatextremes in hypocoagulability protect against ischaemic cardiovasculardisease. A mild decrease in coagulability found in hemophiliac patientscan have a protective effect against fatal ischaemic heart disease(Sramek A, et al. (2003) Lancet 362(9381):351-4; Bilora F, et al. (1999)Clin Appl Thromb Hemost. 5(4):232-5.

The aptamers can be administered to prevent coagulation-inducedinflammation. Inflammation is induced by thrombolytic therapy inpatients with acute myocardial infarction (AMI), which might contributeto microvascular obstruction and reperfusion injury. Improved aptamersof the present invention can inhibit this early inflammatory response.In one embodiment, methods are provided to reduce the early inflammatoryresponse in mammals that are in need thereof by administering theimproved aptamers of the present invention.

The improved aptamers and antidotes of the present invention can be usedto inhibit atherosclerosis. Some adverse events in atherosclerosis areassociated with ruptured plaques, which are a major cause of morbidityand mortality associated with atherosclerosis. In addition toconventional coronary heart disease risk factors, coagulation factor IXactivation peptide and fibrinogen can be positively associated with riskof coronary heart disease (R. Rosenberg et al. (2001) Thromb Haemost 86:41-50; J A Cooper et al. (2000) Circulation 102: 2816-2822). Theintrinsic pathway may significantly enhance thrombogenicity ofatherosclerotic lesions after removal of the endothelial layer andexposure of SMCs and macrophages to blood flow (Ananyeva N M et al.(2002) Blood 99: 4475-4485). In addition, the improved aptamers can alsobe provided to prevent morbidity in mammals suffering from acutecoronary syndrome (ACS) associated with inflammation.

In certain clinical scenarios, the contact pathway becomes the majorpathway for blood clotting. These include surgical procedures where theblood products are removed from the body, such as contact of the bloodwith the cardiopulmonary bypass (CPB) circuit and oxygenator induces aninflammatory state during and post CPB. Genetic epidemiology andprospective clinical studies have linked the magnitude of theinflammatory response during coronary revascularization procedures withmultiple adverse effects of CPB, including renal damage, atrialfibrillation, stroke, gut damage and neuronal damage. The inflammatoryresponse induced by activation of the coagulation pathway is mediated bycoagulation factors Xa and thrombin, which in addition to their role inblood clot formation, are themselves pro-inflammatory and mitogenicsignaling proteins. The improved aptamers can also be administered toprevent adverse effects associated with post-angioplasty restenosis.

Regulating Coagulation with Improved Aptamer-Antidote Pairs

Among the many challenges of treating patients with thrombotic disordersor during a coagulation-inducing event is the potential risk ofhemorrhage associated with anticoagulant drug therapy. The mechanismswhich underlie bleeding risk are complex, but are unquestionably afunction of drug variability (excess anticoagulant effect for the degreeof thrombogenicity or extent of thrombus burden), relatively poorcorrelation between drug concentration and anticoagulant effect,wide-spread compromise of hemostatic barriers (platelet performance,vascular integrity, multiple phases of coagulation) and limited controlof the anticoagulant's behavior.

At least three clinical scenarios exist in which the ability to rapidlyreverse the activity of an antithrombotic or anticoagulant nucleic acidligand is desirable. The first case is when anticoagulant orantithrombotic treatment leads to hemorrhage, including intracranial orgastrointestinal hemorrhage. While identifying safer target proteins mayreduce this risk, the potential for morbidity or mortality from thistype of bleeding event is such that the risk can not be overlooked. Thesecond case is when emergency surgery is required for patients who havereceived antithrombotic treatment. This clinical situation arises in apercentage of patients who require emergency coronary artery bypassgrafts (CABG) while undergoing percutaneous coronary intervention underthe coverage of GPIIb/IIIa inhibitors. Current practice in thissituation is to allow for clearance of the compound (for small moleculeantagonists such as eptifibatide), which may take 2-4 hours, or plateletinfusion (for Abciximab treatment). The third case is when ananticoagulant nucleic acid ligand is used during a cardiopulmonarybypass procedure. Bypass patients are predisposed to post operativebleeding. In each case, acute reversal of the anticoagulant effects of acompound via an antidote (e.g., an oligonucleotide antidote of theinvention targeted to an anticoagulant or antithrombotic nucleic acidligand) allows for improved, and likely safer, medical control of theanticoagulant or antithrombotic compound.

The applicants have discovered improved aptamer-antidote pairs thatprecisely regulate proteins in the blood coagulation cascade. In oneembodiment, the antidotes of the invention are provided to a mammal inneed thereof after the aptamers of the invention to reverse the effectsof the aptamers. Aptamers and aptamer-antidote pairs can be administeredin real time as needed based on various factors, including the progressof the patient, as well as the physician's discretion in how to achieveoptimal therapy. Thus, this invention discloses an improved regulatabletherapeutic regime in the course of nucleic acid ligand therapy forblood coagulation.

Individuals who are undergoing surgery also require the targetedmodulation of coagulation that occurs through the use of the improvedaptamers and antidotes of the present invention. In certain embodiments,the aptamers are administered to patients undergoing general surgery. Incertain other embodiments, the aptamers are administered to patientswith cardiovascular disease, which can include coronary heart disease.The patients can be undergoing treatment including bypass surgery orpercutaneous coronary interventions. The mammals who can be treated withthe aptamers of the present invention can also include patients who havehad physical trauma that requires coagulation therapy.

Preoperative assessment of the patient can identify drug-induced,acquired, or inherited coagulation defects. The main attention inanticoagulant therapy is directed to the perioperative period. Afurther, often overlooked, management strategy in treating majorcoagulopathies is the consideration of the cost and half-lives of thecoagulation factors in individual blood components. Prevention ofbleeding has become possible by manipulation of the control ofcoagulation and inflammatory processes. Additionally, because diagnosisof patients is often difficult, the modulatable improved aptamers andantidote pairs of the present invention are particularly useful inensuring that, in case of incorrect diagnosis and treatment, treatmentcan immediately be disabled. For example, the symptoms of coronaryinfarction can closely mimic those of an acute coronary dissection. SeeScarabeo et al. (2002) Italian Heart Journal 3: 490-494. A diagnosis ofcoronary infarction immediately calls for an anticoagulant, which iscounterindicated in acute coronary dissection. With the improvedaptamer-antidote pairs described herein, a mistake by a health careprovider can readily be reversed.

Agents that restore vascular patency in stroke also increase the risk ofintracerebral hemorrhage (ICH). As Factor IXa is a key intermediary inthe intrinsic pathway of coagulation, targeted inhibition of FactorIXa-dependent coagulation can inhibit microvascular thrombosis in strokewithout impairing extrinsic hemostatic mechanisms that limit ICH. Theimproved aptamers and antidotes of the present invention can be used toinhibit stroke associated with cardiovascular disease and surgery.

Administration

The present method for treating cardiovascular disease in a tissuecontemplates contacting a tissue in which cardiovascular disease isoccurring, or is at risk for occurring, with a composition comprising atherapeutically effective amount of an improved aptamer capable ofbinding a coagulation factor as well as providing an improved antidoteto reverse the effects of the aptamer by administration of an antidote.Thus, the method comprises administering to a patient a therapeuticallyeffective amount of a physiologically tolerable composition containingthe RNA aptamer as well as a method to provide an antidote to reversethe effects of the aptamer by administration of an antidote.

The dosage ranges for the administration of the antidote depend upon theform of the antidote, and can be assessed by a physician or other healthcare provider. Generally, the dosage will vary with the age, condition,sex and extent of the disease in the patient and can be determined byone of skill in the art. The individual physician in the event of anycomplication can also adjust the dosage.

Generally, a therapeutically effective amount is an amount of a antidotesufficient to produce a measurable modulation of the effects of thenucleic acid ligand, including but not limited to acoagulation-modulating amount or an inflammation-modulating amount.

Preferred modes of administration of the improved aptamers of thepresent invention are parenteral, intravenous, intradermal,intra-articular, intra-synovial, intrathecal, intra-arterial,intracardiac, intramuscular, subcutaneous, intraorbital, intracapsular,intraspinal, intrasternal, topical, transdermal patch, via rectal,vaginal or urethral suppository, peritoneal, percutaneous, nasal spray,surgical implant, internal surgical paint, infusion pump or viacatheter. In one embodiment, the agent and carrier are administered in aslow release formulation such as an implant, bolus, microparticle,microsphere, nanoparticle or nanosphere.

The antidotes of the present invention can be preferably administeredparenterally by injection or by gradual infusion over time. Although thetissue to be treated can typically be accessed in the body by systemicadministration and therefore most often treated by intravenousadministration of therapeutic compositions, other tissues and deliverytechniques are provided where there is a likelihood that the tissuetargeted contains the target molecule. Thus, antidotes of the presentinvention are typically administered orally, topically to a vasculartissue, intravenously, intraperitoneally, intramuscularly,subcutaneously, intra-cavity, transdermally, and can be delivered byperistaltic techniques. As noted above, the pharmaceutical compositionscan be provided to the individual by a variety of routes such orally,topically to a vascular tissue, intravenously, intraperitoneally,intramuscularly, subcutaneously, intra-cavity, transdermally, and can bedelivered by peristaltic techniques. Representative, non-limingapproaches for topical administration to a vascular tissue include (1)coating or impregnating a blood vessel tissue with a gel comprising anucleic acid ligand, for delivery in vivo, e.g. by implanting the coatedor impregnated vessel in place of a damaged or diseased vessel tissuesegment that was removed or by-passed; (2) delivery via a catheter to avessel in which delivery is desired; (3) pumping a nucleic acid ligandcomposition into a vessel that is to be implanted into a patient.Alternatively, the nucleic acid ligand can be introduced into cells bymicroinjection, or by liposome encapsulation. Advantageously, nucleicacid ligands of the present invention can be administered in a singledaily dose, or the total daily dosage can be administered in severaldivided doses. Thereafter, the antidote is provided by any suitablemeans to alter the effect of the nucleic acid ligand by administrationof the antidote.

Compositions

The aptamers and antidotes of the invention can be formulated intopharmaceutical compositions that can include, in addition to an improvedaptamer, a antidote or modulator, and a pharmaceutically acceptablecarrier, diluent or excipient. The precise nature of the compositionwill depend, at least in part, on the nature of the improved aptamer andantidote and the route of administration. Optimum dosing regimens can bereadily established by one skilled in the art and can vary with theimproved aptamer, the antidote combination, the patient and the effectsought.

For standard information on pharmaceutical formulations, see Ansel, etal., Pharmaceutical Dosage Forms and Drug Delivery Systems, SixthEdition, Williams & Wilkins, 1995. The therapeutic compositionscomprising aptamers and antidotes of the present invention areconventionally administered intravenously, as by injection of a unitdose, for example. The term “unit dose” when used in reference to atherapeutic composition of the present invention refers to physicallydiscrete units suitable as unitary dosage for the subject, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requireddiluent; i.e. carrier or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for administration are also variable,but are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated.

Pharmaceutically useful compositions comprising an aptamer or antidoteof the present invention can be formulated according to known methodssuch as by the admixture of a pharmaceutically acceptable carrier.Examples of such carriers and methods of formulation can be found inRemington's Pharmaceutical Sciences. To form a pharmaceuticallyacceptable composition suitable for effective administration, suchcompositions will contain an effective amount of the aptamer. Suchcompositions can contain admixtures of more than one aptamers orantidotes.

The effective amount of an improved aptamer of the invention can varyaccording to a variety of factors such as the individual's condition,weight, sex and age. Other factors include the mode of administration.Generally, the compositions will be administered in dosages adjusted forbody weight, e.g., dosages ranging from about 0.1 mg/kg body weight toabout 100 mg/kg body weight. In specific embodiments, the dosages areabout 0.5 mg/kg body weight to 50 mg/kg body weight. In specificembodiments, the dosage is between 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, and 1 mg/kg body weight, and any dosage in between. Specificdosage units can range from 1 ng to 1 g, but are more conventionallyabout 0.01 μg, 0.1 μg, 1 μg, 10 μg, 100 μg, 500 μg, or 1 g or any amountin between.

The effective amount of antibody being delivered to a patient will varyaccording to a variety of factors such as the individual's condition,weight, sex, age and amount of nucleic acid ligand administered. In oneembodiment, the antidote ranges from 0.5-50 mg/kg. In anotherembodiment, the amount of antidote being delivered ranges from 0.5-10,0.5-5, 1-10 or 1-5 mg/kg. In general, the amount of antidote beingdelivered is not less than the amount of aptamer being delivered.Typically, the amount of antidote is from about 1 to about 20 times theamount of aptamer. In certain embodiments, the antidote is about 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 times the amount of aptamer delivered to apatient.

Improved combinations of aptamers and antidotes in pharmaceuticalcompositions are administered in therapeutically effective amounts, thatis, in amounts sufficient to generate a coagulation-modulating response,or in a prophylactically effective amounts, that is in amountssufficient to prevent a coagulation factor from acting in a coagulationcascade. The therapeutically effective amount and prophylacticallyeffective amount can vary according to the modulator. The pharmaceuticalcomposition can be administered in single or multiple doses.

Because the activity of the improved antidotes is lasting, once thedesired level of modulation of the nucleic acid ligand by the antidoteis achieved, infusion of the antidote can be terminated, allowingresidual antidote to clear the human or animal. This allows forsubsequent re-treatment with the nucleic acid ligand as needed.Alternatively, and in view of the specificity of the antidotes of theinvention, subsequent treatment can involve the use of a second,different improved aptamer/antidote pair.

Antidotes synthesized or identified according to the methods disclosedherein can be used alone at appropriate dosages defined by routinetesting in order to obtain optimal modulation of nucleic acid ligandactivity in coagulation, while minimizing any potential toxicity. Inaddition, co-administration or sequential administration of other agentscan be desirable. For combination treatment with more than one activeagent, where the active agents are in separate dosage formulations, theactive agents can be administered concurrently, or they each can beadministered at separately staggered times.

The dosage regimen utilizing the improved aptamers and antidotes of thepresent invention is selected in accordance with a variety of factorsincluding type, species, age, weight, sex and medical condition of thepatient; the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular combination employed. A physician of ordinary skill canreadily determine and prescribe the effective amount of the aptamerrequired to prevent, counter or arrest the progress of the condition.Optimal precision in achieving concentrations of the combination withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the aptamer and antidote's availability to targetsites. This involves a consideration of the distribution, equilibrium,and elimination of the modulator.

In the methods of the present invention, the combinations described indetail can form the active ingredient, and are typically administered inadmixture with suitable pharmaceutical diluents, excipients or carriers(collectively referred to herein as “carrier” materials) suitablyselected with respect to the intended form of administration, that is,oral tablets, capsules, elixirs, syrup, suppositories, gels and thelike, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders includewithout limitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include, without limitation, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitablyflavored suspending or dispersing agents such as the synthetic andnatural gums, for example, tragacanth, acacia, methyl-cellulose and thelike. Other dispersing agents that can be employed include glycerin andthe like. For parenteral administration, sterile suspensions andsolutions are desired. Isotonic preparations that generally containsuitable preservatives are employed when intravenous administration isdesired.

Topical preparations containing the active drug component can be admixedwith a variety of carrier materials well known in the art, such as,e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and Eoils, mineral oil, PPG2 mydstyl propionate, and the like, to form, e.g.alcoholic solutions, topical cleansers, cleansing creams, skin gels,skin lotions, and shampoos in cream or gel formulations.

The aptamers and antidotes of the present invention can also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids, suchas cholesterol, stearylamine or phosphatidylcholines.

The aptamers and antidotes of the present invention can also be coupledwith soluble polymers as targetable drug carriers. Such polymers caninclude polyvinyl-pyrrolidone, pyran copolymer,polyhydroxypropylmethacryl-amidephenol,polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the aptamers andantidotes of the present invention can be coupled (preferably via acovalent linkage) to a class of biodegradable polymers useful inachieving controlled release of a drug, for example, polyethylene glycol(PEG), polylactic acid, polyepsilon caprolactone, polyhydroxy butyricacid, polyorthoesters, polyacetals, polydihydro-pyrans,polycyanoacrylates and cross-linked or amphipathic block copolymers ofhydrogels. Cholesterol and similar molecules can be linked to theaptamers to increase and prolong bioavailability.

In certain embodiments of this invention, the complex comprises aliposome with a targeting nucleic acid ligand (s) associated with thesurface of the liposome and an encapsulated therapeutic or diagnosticagent. Preformed liposomes can be modified to associate with the nucleicacid ligands. For example, a cationic liposome associates throughelectrostatic interactions with the nucleic acid. Alternatively, anucleic acid attached to a lipophilic compound, such as cholesterol, canbe added to preformed liposomes whereby the cholesterol becomesassociated with the liposomal membrane. Alternatively, the nucleic acidcan be associated with the liposome during the formulation of theliposome. Preferably, the nucleic acid is associated with the liposomeby loading into preformed liposomes.

EXAMPLES Tests of Aptamers for Coagulation

The following tests are used to assess the capacity of modified aptamersand antidotes to inhibit coagulation factors.

The Activated Clotting Time Test (ACT) is a screening test thatresembles the activated partial thromboplastin time (APTT) test, but isperformed using fresh whole blood samples. ACT can be to monitor apatient's coagulation status in connection with clinical procedures,such as those that involve the administration of high doses of heparin(e.g., CPB and PTCA).

The Activated Partial Thromboplastin Time Test (APTT) is a commoncentral laboratory test, typically performed using an automatedcoagulometer, for example Diagnostica Stago's STA coagulometer(MDA/96/23), or another coagulometer produced by this company orotherwise known in the art. The test is performed using a plasma sample,in which the intrinsic pathway is activated by the addition ofphospholipid, an activator (ellagic acid, kaolin, or micronized silica),and Ca²⁺.

The bleeding time test can be used for the diagnosis of hemostaticdysfunction, von Willebrand's disease, and vascular disorders. It alsocan be used to screen for platelet abnormalities prior to surgery. Thetest is performed by making a small incision on the forearm and wickingaway the blood from the wound site. The time it takes for bleeding tostop is recorded and in control subjects is approximately 3.5 minutes.Prolongation of the bleeding time is indicative of qualitative orquantitative platelet defects.

The Prothrombin Time Test (PT), which was first described by Quick in1935, measures the tissue factor-induced coagulation time of blood orplasma. It is used as a screening test to evaluate the integrity of theextrinsic coagulation pathway, and is sensitive to coagulation factorsI, II, V, VII, and X. The test can be performed by adding thromboplastinand Ca²⁺ to a patient sample and measuring the time for clot formation.A prolonged clotting time suggests the presence of an inhibitor to, or adeficiency in, one or more of the coagulation factors of the extrinsicpathway. But PT clotting time can also be prolonged for patients onwarfarin therapy, or for those with vitamin K deficiency or liverdysfunction. The PT test can provide an assessment of the extrinsiccoagulation pathway, and is widely used to monitor oral anticoagulationtherapy.

The Thrombin Clotting Time Test (TCT) measures the rate of a patient'sclot formation compared to that of a normal plasma control. The test canbe performed by adding a standard amount of thrombin to a patient'splasma that has been depleted of platelets, and measuring the timerequired for a clot to form. This test has been used as an aid in thediagnosis of disseminated intravascular coagulation (DIC) and liverdisease.

There are also a number of tests that may be used in the diagnosis of apatient's coagulative status. These fall into two categories: complextests, some of which are based on the screening tests outlined above,and immunoassays. Complex Tests include specific factor assays based onlaboratory tests, such as the APTT, PT, and TCT tests. One assaymeasures the level of the activation Factor IXa or the FactorIXa-antithrombin III complex. These measurements are used to determinethe levels of factor IXa or factor VII-tissue mediated complex. Assaysfor activated protein C resistance, antithrombin, protein C deficiency,and protein S deficiency are also part of this group. Asymptomaticindividuals who have heterogeneous deficiencies of proteins C and S, andresistance to activated protein C, have significantly elevated levels ofthe prothrombin fragment F1.2 compared to controls.

Example 1 Substitution of 2′-O-methyl for 2′-O-methyl for 2′-hydroxylSugars in Sectors

2′-Hydroxyl purines were substituted with 2′-O-methyl purines in the 4secondary structure units of in which purine residues are present: Stem1 (Apt 1); Loop 1 (Apt 2); Stem 2 (Apt 3); Loop 2 (Apt 4) (see FIG. 1A).

Procedure: The anticoagulant activity of AptA derivatives Apt 1-5 wasevaluated in standard APTT coagulation assays over compoundconcentrations ranging from 1 uM to low nanomolar (FIG. 2). The“neutralizability” of Apt1-5 was evaluated in standard APTT antidoteassays over AptA antidote concentrations (AptA AD; see sequencelistings) ranging from 5 uM and down (FIG. 2). For these assays, theconcentration of AptA and derivatives was fixed at 125 nM.

Apt 4 showed gain of anticoagulant activity (FIG. 2); Apt 1-3 showedmoderate loss of activity; and Apt 5 showed severe loss of activity. Apt1-3 exhibit enhanced neutralization, suggesting that introduction of2′-O-methyl residues within the antidote binding site improves theability of the antidote oligonucleotide to bind to the aptamer.

Sequence Listings:

1) AptA Length: 35 (5′-3′) sequence:AUGGGGA CUAUACC GCG UAAUGC UGC C UCCCCAU T (SEQ ID NO: 20) 1) Apt 1Length: 35 (5′-3′) sequence: aUgggga CUAUACCGCGUAAUGCUGCC UCCCCaU T(SEQ ID NO: 21) 3) Apt 2 Length: 35 (5′-3′) sequence:AUGGGGA CUaUaCC GCG UAAUGC UGC C UCCCCAU T (SEQ ID NO: 22) 4) Apt 3Length: 35 (5′-3′) sequence: AUGGGGA CUAUACC gCg UAAUGC UgC C UCCCCAU T(SEQ ID NO: 23) 5) Apt 4 Length: 35 (5′-3′) sequence:AUGGGGA CUAUACC GCG UaaUgC UGC C UCCCCAU T (SEQ ID NO: 24) 6) Apt 5Length: 35 (5′-3′) sequence: aUgggga CUaUaCC gCg UaaUgC UgC C UCCCCaU T(SEQ ID NO: 25) 7) AptA AD Length: 17 (5′-3′) sequence:cgcgguauaguccccau (SEQ ID NO: 1) “A”: 2′OH Adenine; “a”: 2′-O-methylAdenine; “G”: 2′OH Guanine; “g”: 2′-O-methyl Guanine; “C”:2′Fluro-Cytidine; “c”: 2′-O-methyl Cytidine; “U”: 2′Fluoro-Uridine; “u”:2′-O-methyl Uridine; “T”: inverted 2′H Thymidine.

Example 2 Stem 1 Modifications

Two “families” of stem 1 variants were designed (Apt 6-8 and 9-11; FIG.1B) consisting of 4, 5, and 6 basepair stems. All constructs weredesigned in the Apt-2 background. Stem 1 sequences were evaluated forthe ability to design complementary antidote oligonucleotides to themsuch that the antidotes contain minimal secondary structure, and for theability of the aptamer to assume the proper secondary structure.

Stems were wholly 2′-O-methyl modified. Antidote oligonucleotides weredesigned specific for Apt 6-11 that bind to their respective targetaptamer in the same register as AptA AD (see sequence listings below).

Experiments: The anticoagulant activity of Apt 6-11 was evaluated instandard APTT coagulation assays over compound concentrations rangingfrom 1 uM to low nanomolar. The antidote control of Apt 6-11 wasevaluated in standard APTT antidote assays over antidote concentrationsranging from 5 uM and down. For these assays, the concentration of Apt2, and Apt 6-11 was set at 250 nM (as opposed to 125 nM for standardAptA and Apt 2 experiments).

Apt 6-8 exhibit loss of anticoagulant activity (FIG. 3), however, allexhibit similar activity levels. Thus stem length is not be the maincause for loss of activity. The 5 base pair stem 1 constructs (Apt 10and Apt 7) do appear to be more neutralizable than Apt 2 (FIGS. 3 and4). Data suggests that a stem 1 of 5 base pairs may be preferable tothose composed of 4, 6 or 7 base pairs to enhance antidoteneutralization.

Sequence Listings:

1) Apt 6 Length: 29 (5′-3′): ggga CUaUaCCGCGUAAUGCUGCC uccc T(SEQ ID NO: 26) 2) Apt 7 Length: 31(5′-3′): gugga CUaUaCCGCGUAAUGCUGCC uccac T (SEQ ID NO: 27) 3) Apt 8Length: 33 (5′-3′): gaugga CUaUaCCGCGUAAUGCUGCC uccauc T (SEQ ID NO: 28)4) Apt 9 Length: 29 (5′-3′): cuga CUaUaCCGCGUAAUGCUGCC ucag T(SEQ ID NO: 29) 5) Apt 10 Length: 31(5′-3′): ccuga CUaUaCCGCGUAAUGCUGCC ucagg T (SEQ ID NO: 30) 6) Apt 11Length: 33 (5′-3′): cucuga CUaUaCCGCGUAAUGCUGCC ucagag T (SEQ ID NO: 31)7) Apt 6 AD Length: 14 (5′-3′): cgcgguauaguccc (SEQ ID NO: 2)8) Apt 7 AD Length: 15 (5′-3′): cgcgguauaguccac (SEQ ID NO: 3)9) Apt 8 AD Length: 16 (5′-3′): cgcgguauaguccauc (SEQ ID NO: 4)10) Apt 9 AD Length: 14 (5′-3′): cgcgguauagucag (SEQ ID NO: 5)11) Apt 10 AD Length: 15 (5′-3′): cgcgguauagucagg (SEQ ID NO: 6)12) Apt 11 AD Length: 16 (5′-3′): cgcgguauagucagag (SEQ ID NO: 7) “A”:2′OH Adenine; “a”: 2′-O-methyl Adenine; “G”: 2′OH Guanine; “g”:2′-O-methyl Guanine; “C”: 2′Fluro-Cytidine; “c”: 2′-O-methyl Cytidine;“U”: 2′Fluoro-Uridine; “u”: 2′-O-methyl Uridine; “T”: inverted 2′HThymidine.

Example 3 Stem 1 Sugar Chemistry

The anticoagulant activity of Apt 12-17 was evaluated in standard APTTcoagulation assays over compound concentrations ranging from 1 uM to lownanomolar. The “neutralizability” of Apt 12-17 was evaluated in standardAPTT antidote assays over antidote concentrations ranging from 5 uM anddown. For Apt 12, 14, 15, and 16, the aptamer concentration was fixed at125 nM in these assays, and for Apt 13 and 17, the aptamer concentrationwas fixed at 250 nM.

Comparison of the anticoagulant activity of Apt 12 with Apt 13 and Apt17(FIG. 5) demonstrates that the loss of activity observed for Apt6-11 isdue to the presence of 2′-O-methyl substitutions at one or more criticalresidues. Comparison of the anticoagulant activity of Apt14 to Apt12indicates that the stretch of 4 consecutive guanosines within stem 1 canbe altered without a significant impact on anticoagulant activity.Comparison of Apt15 and 16 with Apt 2, 12 and 17 a) demonstrates thatthe presence of 2′-O-methyl sugars at each position within stem 1 exceptfor the closing A-U pair at the top of stem 1 enhances activity; b)demonstrates that the sugar of the U in this base pair must be 2′ fluorofor the aptamer to retain potency; and c) suggests that the sugar of theA in this base pair can be a 2′-O-methyl sugar without a significantimpact on anticoagulant activity. In fact, Apt 16 retains essentiallyfull potency.

Comparison of the neutralization of Apt14-16 with Apt14/AD suggests thatthe antidote can more readily bind the aptamer when stem 1 is a2′-O-methyl-2′ fluoro stem as opposed to when both strands of the duplexcontain largely 2′-O-methyl residues. Apt21 was designed in which thesugar of the A at the stop of stem 1 is 2′-O-methyl substituted (FIG.1). Substitution of a 2′-O-methyl sugar at this adenosine residue iswell tolerated in the background of a largely 2′-O-methyl stem (FIG. 6).Antidote neutralization of Apt 21 is enhanced as compared to Apt16 (seeespecially the 2.5:1 and 5:1 AD:Drug data points in FIG. 4).

Sequence Listings:

1) Apt2 Length: 35 (5′-3′) sequence:aUgggga CUaUaCCGCGUAAUGCUGCC UCCCCaU T (SEQ ID NO: 32) 2) Apt13Length: 35 (5′-3′) sequence: augggga CUaUaCCGCGUAAUGCUGCC uccccau T(SEQ ID NO: 33) 3) Apt14 Length: 35 (5′-3′) sequence:gUgagga CUaUaCCGCGUAAUGCUGCC UCCUCaC T (SEQ ID NO: 34) 4) Apt15Length: 35 (5′-3′) sequence: gUgaggA CUaUaCCGCGUAAUGCUGCC UCCUCaC T(SEQ ID NO: 35) 5) Apt16 Length: 35 (5′-3′) sequence:gugaggA CUaUaCCGCGUAAUGCUGCC Uccucac T (SEQ ID NO: 36) 6) Apt17Length: 35 (5′-3′) sequence: gugagga CUaUaCCGCGUAAUGCUGCC uccucac T(SEQ ID NO: 37) 7) Apt14AD Length: 17(5′-3′) sequence: cgcgguauaguccucac (SEQ ID NO: 8) 8) Apt21 Length: 35(5′-3′) sequence: gugagga CUaUaCC GCG UAAUGC UGC C Uccucac T(SEQ ID NO: 41) “A”: 2′OH Adenine; “a”: 2′-O-methyl Adenine; “G”: 2′OHGuanine; “g”: 2′-O-methyl Guanine; “C”: 2′Fluro-Cytidine; “c”:2′-O-methyl Cytidine; “U”: 2′Fluoro-Uridine; “u”: 2′-O-methyl Uridine;“T”: inverted 2′H Thymidine.

Example 4 Reducing Length of Stem 1

The anticoagulant activity of Apt 18-20 was evaluated in standard APTTcoagulation assays over compound concentrations ranging from 1 uM to lownanomolar. The “neutralizability” of Apt 18-20 was evaluated in standardAPTT antidote assays over antidote concentrations (Antidote 6, 7 and 8for 18-20 respectively) ranging from 5 uM and down. The aptamerconcentration was fixed at 125 nM in these assays.

Each of the aptamers (Apt 18-20) is a potent anticoagulant, as or morepotent than Apt 2 (FIG. 7). Furthermore, all three are readilyneutralized by their respective antidote oligonucleotides. Apt 19 wasevaluated for anticoagulant activity of a pegylated version. PEG Apt 19and, for comparison, PEG-Apt 16 are used (PEG is a 40 KDa polyethyleneglycol mPEG2-NHS ester (MW 40 kDa; Nektar/Shearwater 2Z3XOT01), appendedto the 5′ end via conjugation to a C6 amino linker added to the aptamerduring solid phase synthesis). FIG. 8 indicates that the length of stem1 does not affect how 40 KDa PEG addition impacts the activity of AptAand AptA derivatives. The anticoagulant activity of PEG Apt 19 and 16 isessentially identical to the anticoagulant activity of both pegylated(PEG AptA) and cholesterol-modified (CH-AptA) versions of the parentalAptA sequence. In addition, like Apt 19, PEG Apt 19 is more readilyneutralized by its matched antidote (7 AD) than AptA, Apt 16 or any ofthe PEG or cholesterol-modified versions of these compounds. FIG. 8shows approximately 90% reversal of PEG Apt 19 at 2.5:1 AD:Aptamer.Looked at as an absolute change in APTT rather than on a % reversalbasis, the APTT of plasma treated with PEG Apt 19+2.5:1 7AD:Aptamer isonly 4-5 seconds above baseline.

Sequence Listings:

1) Apt18 Length: 29 (5′-3′) sequence: gggA CUaUaCCGCGUAAUGCUGCC Uccc T(SEQ ID NO: 38) 2) Apt19 Length: 31 (5′-3′) sequence:guggA CUaUaCCGCGUAAUGCUGCC Uccac T (SEQ ID NO: 39) 3) Apt20 Length: 33(5′-3′) sequence: gauggA CUaUaCCGCGUAAUGCUGCC Uccauc T (SEQ ID NO: 40)4) PEG Apt 16 Length: 35 (5′-3′) sequence: P-L-gugaggACUaUaCCGCGUAAUGCUGCC Uccucac T 5) PEG Apt 19 Length: 31(5′-3′) sequence: P-L-guggA CUaUaCCGCGUAAUGCUGCC Uccac T “A”: 2′OHAdenine; “a”: 2′-O-methyl Adenine; “G”: 2′OH Guanine; “g”: 2′-O-methylGuanine; “C”: 2′Fluro-Cytidine; “c”: 2′-O-methyl Cytidine; “U”:2′Fluoro-Uridine; “u”: 2′-O-methyl Uridine; “T”: inverted 2′H Thymidine;“P”: mPEG2-NHS ester MW 40 kDa (Nektar/Shearwater 2Z3XOT01); “L”: C6amino linker

Example 5 Stem 2 and Loop 2 Substitutions

Two series of variants evaluating the optimal sugar composition for theresidues in stem 2 and loop 2. The first series in the Apt 16background. The second series in the Apt 16 background, but substitutingthe tetraloop found in FIXa aptamer 9.20 (see Rusconi et al Nature 419,p. 90-94, 2002 and FIG. 1) for the hexanucleotide loop found in AptA.Studies on Apt 4 indicated that 2′-O-methyl purine substitution withinloop 2 led to an enhancement in AptA potency, whereas 2′-O-methyl purinesubstitution within stem 2 led to a modest loss of potency, and thatsimultaneous 2′-O-methyl purine substitution within stem 2 and loop 2 inthe context of a 2′-O-methyl purine stem 1 led to a significant loss ofAptA potency (Apt 5). Therefore, independently substitute 2′-O-methylpurines in stem 2 (Apt 22, 26) and loop 2 (Apt 23, 27) (FIG. 9).Re-evaluated complete 2′-O-methyl substitution of purines within stem 2and loop 2 (Apt 24, Apt 28) but leave the G at the base of stem 2 as a2′ hydroxyl (Apt 25, 29) in the event that a 2′ hydroxyl is required atthis position.

The anticoagulant activity of Apt 22-29 was evaluated in standard APTTcoagulation assays over compound concentrations ranging from 1 uM to lownanomolar. The “neutralizability” of Apt 22-29 was evaluated in standardAPTT antidote assays over antidote concentrations ranging from 5 uM anddown. The aptamer concentration was fixed at 125 nM in these assays,except for Apt 24 in which the aptamer concentration was fixed at 250nM.

As previously observed with Apt 3, substitution of 2′-O-methyl purineswithin loop 2 leads to enhanced potency (Apt 23, compare Apt 23 to 16)(FIG. 9). Likewise, substitution of 2′-O-methyl purines into stem 2leads to a moderate loss of activity (Apt 22, 24) (FIG. 14). Apt 24 issignificantly more potent than Apt 5. Maintenance of a 2′ hydroxyl onthe G residue at the base of stem 2 (Apt 25) does not lead to enhancedactivity as compared to Apt 24, indicating that a) substitution of a2′-O-methyl sugar at this residue is not the problem within Apt 22 and24 and b) the sugar on this residue can be 2′-O-methyl. Substitution ofthe 9.20 tetraloop for the hexanucleotide loop present in AptA led to aloss of activity (Apt 26-29). Antidote neutralization of Apt 23 isreduced as compared to Apt 16, but still equivalent to AptA.

Sequence Listings:

1) Apt22 Length: 35 (5′-3′) sequence:gugaggA CUaUaCC gCg UAAUGC UgC C Uccucac T (SEQ ID NO: 42) 2) Apt23Length: 35 (5′-3′) sequence: gugaggA CUaUaCC GCG UaaUgC UGC C Uccucac T(SEQ ID NO: 43) 3) Apt24 Length: 35 (5′-3′) sequence:gugaggA CUaUaCC gCg UaaUgC UgC C Uccucac T (SEQ ID NO: 44) 4) Apt25Length: 35 (5′-3′) sequence: gugaggA CUaUaCC GCg UaaUgC UgC C Uccucac T(SEQ ID NO: 45) 5) Apt26 Length: 33 (5′-3′) sequence:gugaggA CUaUaCC gCa AUCG UgC C Uccucac T (SEQ ID NO: 46) 6) Apt27Length: 33 (5′-3′) sequence: gugaggA CUaUaCC GCA aUCg UGC C Uccucac T(SEQ ID NO: 47) 7) Apt28 Length: 33 (5′-3′) sequence:gugaggA CUaUaCC gCa aUCg UgC C Uccucac T (SEQ ID NO: 48) 8) Apt29Length: 33 (5′-3′) sequence: gugaggA CUaUaCC GCa aUCg UgC C Uccucac T(SEQ ID NO: 49)

Example 6 Stem 2 Sugar Chemistry

The anticoagulant activity of Apt 30-33 was evaluated in standard APTTcoagulation assays over compound concentrations ranging from 1 uM to lownanomolar. The “neutralizability” of Apt 30 and 33 was evaluated instandard APTT antidote assays over antidote concentrations (Apt 14 AD)ranging from 5 uM and down. The aptamer concentration was fixed at 125nM in these assays (see FIG. 10).

Comparison of the activity of Apt 30 and 33 to Apt 31 and 32demonstrates that C16 needs to contain a 2′ fluoro sugar and G25 a 2′hydroxyl sugar (FIG. 10 a). Activity observed between Apt 31 and 32suggests that remaining positions within stem 2 can contain 2′-O-methylsugars. In fact, Apt 31 appears to possess slightly greater potency thanApt 32, indicating that a compound with 2′ fluoro at C16, 2′ hydroxyl atG25, and the remaining residues 2′-O-methyl may exhibit greater potencythan Apt 33. Regardless, Apt 33 exhibits greater activity than Apt 2 andis fairly equivalent to original AptA. Apt 33 is more readilyneutralizable than Apt 30, suggesting additional 2′-O-methyl residuewithin the antidote-binding site of the aptamer improves antidotebinding.

Apt 34 had C16 a 2′ fluoro rather than 2′-O-methyl (FIG. 10 b). Increasein anticoagulant activity (compare Apt 34 to Apt 33). However,substitution did result in a modest loss of “neutralizability”, although34 still requires a lower excess of antidote to achieve 90%neutralization (˜5:1 vs 10:1) than the parental AptA compound. Bothresults are consistent with an increase in the stability of stem 2 dueto 2′-O-methyl substitution.

Sequence Listings:

1) Apt30 Length: 35 (5′-3′) sequence:gugagga CUaUaCC gCG UaaUgC UGC C Uccucac T (SEQ ID NO: 50) 2) Apt31Length: 35 (5′-3′) sequence: gugagga CUaUaCC gcg UaaUgC ugc C Uccucac T(SEQ ID NO: 51) 3) Apt32 Length: 35 (5′-3′) sequence:gugagga CUaUaCC gcg UaaUgC UgC C Uccucac T (SEQ ID NO: 52) 4) Apt33Length: 35 (5′-3′) sequence: gugagga CUaUaCC gCg UaaUgC UGC C Uccucac T(SEQ ID NO: 53) 5) Apt34 Length: 35 (5′-3′) sequence:gugagga CUaUaCC gCg UaaUgC uGc C Uccucac T (SEQ ID NO: 54)

Example 7 Individual Base Modifications

Apt35-39 compared with original AptA (numbering based upon AptA stem 1):

-   -   1) Apt 30 to 31: differences are C16, G17, U24, G25, C26.    -   2) Apt 30 to 32: differences are C16, G17, G25.    -   3) Apt 31 to 32: differences are U24, C26.

The anticoagulant activity of Apt 35-39 was evaluated in standard APTTcoagulation assays over compound concentrations ranging from 1 uM to lownanomolar. The “neutralizability” of Apt 35, 38 and 39 was evaluated instandard APTT antidote assays over antidote concentrations (Apt 7 AD)ranging from 5 uM and down. The aptamer concentration was fixed at 125nM in these assays (FIG. 11).

Apt 35-39: Anticoagulant activity of Apt 39 is superior to Apt 19 andall other stem 2-optimization constructs in the Apt 19 background (FIG.11). Results were consistent with those obtained with Apt 34. Inaddition, potency of Apt 39 is comparable to parental AptA and greaterthan Apt 2. The neutralization of Apt 39 by Apt7AD is excellent, andsimilar to neutralization of Apt 19 (FIG. 12). Again, sugar optimizationand truncation of Apt 39 has resulted in a compound neutralized at lowerexcesses of antidote:drug as compared to parental AptA and Apt 2 (FIG.11).

Sequence Listings:

1) Apt35 Length: 31 (5′-3′) sequence:gugga CUaUaCC gCG UaaUgC UGC C Uccac T (SEQ ID NO: 55) 2) Apt36Length: 31 (5′-3′) sequence: gugga CUaUaCC gCG UaaUgC ugc C Uccac T(SEQ ID NO: 56) 3) Apt37 Length: 31 (5′-3′) sequence:gugga CUaUaCC gCG UaaUgC UgC C Uccac T (SEQ ID NO: 57) 4) Apt38Length: 31 (5′-3′) sequence: gugga CUaUaCC gCg UaaUgC UGC C Uccac T(SEQ ID NO: 58) 5) Apt39 Length: 31 (5′-3′) sequence:gugga CUaUaCC gCg UaaUgC uGc C Uccac T (SEQ ID NO: 59)

Example 8 Conjugation of Aptamer to Delivery Vehicle

The anticoagulant tested is Apt39 with a 40 KDa polyethylene glycol(PEG) conjugated to the 5′ end of the aptamer sequence via a 6-carbonNH₂ linker (PEG-Apt39). The antidote is Apt7AD.

The anticoagulant activity of PEG-Apt39 was evaluated in standard APTTcoagulation assays over compound concentrations ranging from 1 uM to lownanomolar. The anticoagulant activity of Apt39 was compared to twoformulations of the parental AptA, CH-Apt S (5′ cholesterol-modified)and PEG-AptA (5′ 40 KDa PEG-modified). For these studies, the molecularweight of the “aptamer” portion only was used to calculate theconcentration of each compound. The “neutralizability” of PEG-Apt39 wasevaluated in standard APTT antidote assays over antidote concentrations(Apt7AD) ranging from 5 uM and down. The aptamer concentration was fixedat 125 nM in these assays.

The in vitro anticoagulant activity of PEG-Apt39 is essentiallyequivalent to CH-AptA and PEG-AptA (FIG. 13).

In Vivo Studies

This study compares the in vivo anticoagulant and antidoteneutralization activity of PEG-Apt39 in swine to previous data obtainedwith CH-AptA with respect to: a) Potency and durability of anticoagulantactivity and the b) Neutralization of anticoagulant activity. The threeexperimental groups (n=2 animals for each) are a) Systemicanticoagulation; b) Systemic anticoagulation and drug neutralization;and c) Systemic anticoagulation, drug neutralization andre-anticoagulation.

Experiment: Six neonatal piglets (1 week old, 2.5-3.5 kg) were randomlyassigned to three groups. Femoral arterial and venous lines were placedin the piglet. The arterial line was used to monitor blood pressure andarterial blood sampling. The venous line was used to administer drugsand test compounds as specified. Temperature of the piglet was monitoredwith a nasopharyngeal temperature probe.

The dose of PEG-Apt39 was 0.5 mg/kg (dose of aptamer based uponmolecular weight of nucleic acid component only; 10,103.2 Da) for eachanimal. In the prior experiments with CH-AptA, the aptamer dose was also0.5 mg/kg (dose of aptamer based upon molecular weight of nucleic acidcomponent only). In experiments in which Apt7 AD was used as anantidote, the dose of the antidote was 3 mg/kg. By comparison, inexperiments with CH-AptA in which AptA AD was used, the antidote dosewas 5 mg/kg.

a) Systemic anticoagulation. A pre-injection blood sample was takenprior to injection of PEG-Apt39, the drug was then injected (time ofinjection is t=0), and blood samples removed at 5, 15, 25, 60, 90, 120and 150 minutes post injection. Activated clotting times (ACT's) wereperformed on-site immediately after blood draw on the whole blood induplicate using the Hemochron 801 junior and glass-activated flip-toptubes per the manufacturers directions. Blood samples were thentransferred to citrated vacutainer tubes and stored on ice. Plateletpoor plasma was prepared, and APTT and PT assays performed per thestandard protocol. (FIG. 14A)

The in vivo anticoagulant potency of PEG-Apt39 is superior to CH-AptA.In addition, the loss of anticoagulant activity over time is reduced forPEG-Apt39 vs. CH-AptA. These results are in contrast with the in vitroanticoagulant activity studies, which demonstrate that the anticoagulantactivity of PEG-Apt39 and CH-AptA are equivalent in vitro in pooledhuman plasma.

b) Systemic anticoagulation and drug neutralization. A pre-injectionblood sample was taken prior to injection of PEG-Apt39, the drug wasthen injected (0.5 mg/kg; time of injection is t=0), and blood samplesremoved at 5 and 15 minutes post drug injection. At t=15 minutes postdrug injection, REG1 S7 AD was administered (3 mg/kg), and additionalblood samples removed at 25, 60, 90, 120 and 150 minutes post druginjection. Activated clotting times (ACT's) were performed on-siteimmediately after blood draw on the whole blood in duplicate using theHemochron 801 junior and glass-activated flip-top tubes per themanufacturers directions. Blood samples were then transferred tocitrated vacutainer tubes and stored on ice. Platelet poor plasma wasprepared, and APTT and PT assays performed per the standard protocol.

Essentially complete neutralization of the anticoagulant activity ofPEG-Apt39 was achieved within 10 minutes of administration of 3 mg/kgApt7 AD. The anticoagulant activity remained neutralized throughout theremainder of the experiment (2 hr and 5 min after initial demonstrationof drug neutralization). Thus, the neutralization of PEG-Apt39 appearsto be superior to that of CH-AptA, as similar levels of neutralizationof PEG-Apt39 can be achieved with a 40% lower dose of antidote (3 mg/kgof Apt7 AD vs. 5 mg/kg REG1 AD). This in vivo data is consistent with invitro experiments in pooled human plasma in which PEG-Apt39 is morereadily neutralized by its matched antidote than any prior formulationof AptA. (FIG. 14B)

c) Systemic anticoagulation, drug neutralization and re-anticoagulation.A pre-injection blood sample was taken prior to injection of PEG-Apt39,the drug was then injected (0.5 mg/kg; time of injection is t=0), andblood samples removed at 5 and 15 minutes post drug injection. At t=15minutes post drug injection, Apt7 AD was administered (3 mg/kg), andadditional blood samples removed at 25, and 40 minutes post druginjection. At t=45 minutes post drug injection (30 minutes followingantidote administration), PEG-Apt39 was re-administered (0.5 mg/kg) andadditional blood samples removed at 50, 60, 90, 120, and 150 minutespost drug injection. Activated clotting times (ACT's) were performedon-site immediately after blood draw on the whole blood in duplicateusing the Hemochron 801 junior and glass-activated flip-top tubes perthe manufacturers directions. Blood samples were then transferred tocitrated vacutainer tubes and stored on ice. Platelet poor plasma wasprepared, and APTT and PT assays performed per the standard protocol.(FIG. 15)

Re-administration of PEG-Apt39 following neutralization of the initialdrug dose is feasible within 30 minutes of administration of theneutralizing antidote. The levels of anticoagulation achieved followingadministration of the first and second dose appear to be equivalent toeach other, suggesting that there is little remaining “free” antidote inthe circulation at the time of administration of the second dose ofdrug.

Example 9 Quantification of Aptamer Complex Formation in Plasma

Aptamer levels in plasma are determined using a sandwich-typehybridization assay with an enzyme-linked immunoassay (ELISA) fordetection. Quantitation of aptamer employs two oligonucleotide probes, aDNA capture probe, and a 2′Omethyl RNA detection probe. The DNA captureprobe is 15 nucleotides in length, is complementary to the 3′ terminal15 nucleotides of the aptamer, and contains a biotin moiety on its 5′terminus, allowing for capture of oligonucleotide complexes containingthis probe to an avidin coated surface. The 2′Omethyl RNA detectionprobe is also 15 nucleotides in length, is complementary to the portionof aptamer to which antidote binds, and contains a digoxigenin moiety toenable detection of complexes containing this probe using standardenzyme-linked fluorescence generating enzyme/substrate reagents.

Quantitation of aptamer is achieved by hybridization of the capture anddetection probes to aptamer in plasma and subsequent immobilization ofthe complex onto the surface of a Neutravidin-coated microtitre plate byway of the 5′-biotin group. Measurement of the digoxigenin-labeled2′-O-methyl RNA probe is performed subsequent to the plateimmobilization reaction using an anti-digoxigenin antibody conjugated toalkaline phosphatase, which catalyzes the fluorescence of a substrate.Fluorescence intensity is then measured, the signal of which is directlyproportional to the amount of aptamer present in the calibrationstandards and validation samples.

The in vitro anticoagulant activity of aptamer Apt39 (SEQ ID NO:88) inplasma from cynomolgus monkeys is reflected by concentration-dependentprolongation of time-to-clot in the APTT assay. Plasma FIX assays wereperformed to aid in interpretation of the Apt39 APTT dose-response curvein monkey plasma. As shown in Table A, the APTT in monkey plasma issensitive to the FIX level. However, the magnitude of the response toreduction in the FIX level is modest. A 75% reduction in the FIX levelresults in a 1.4-fold increase in the APTT, a >95% reduction in the FIXlevel results in a doubling of the APTT, and a 99.9% reduction in theplasma FIX level yields a 2.5-fold increase in the APTT.

TABLE A FIX Activity Assay Standard Curve in Cynomolgus Monkey Plasma %FIX Level APTT Clot Time Fold Increase in Clot Time 100*    35.1 1.050    41.9 1.2 25    49.4 1.4 12.5  55.9 1.6 6.25 62.2 1.8 3.13 68.0 1.91.56 74.7 2.1 0.78 77.7 2.2 0.39 83.8 2.4  0.098 88.1 2.5 *100% FIXlevel represents a 1:5 dilution of normal pooled cynomolgus plasma inbuffer. Human FIX-deficient plasma (George King Biomedical) was used asthe source of FIX-deficient plasma.The date in table A indicate that ˜6 μg/mL Apt39 is required to inhibitapproximately 90% of plasma FIX activity in monkeys (i.e., thisconcentration yields a 1.6-fold increase in the APTT), and that >95%inhibition of plasma FIX activity occurs at Apt39 concentrations of10-12 μg/mL.In Vivo Activity of Apt39 and Apt7AD in Cynomolgus Monkeys

The relationship between the anticoagulant properties of Apt39 and theApt39/Apt7AD complex and the plasma levels of these compounds wasevaluated in monkey. Briefly, 12 monkeys were assigned to threetreatment groups. Group 1 received the anti-FIXa aptamer Apt39, Group 2received the antidote Apt7AD and Group 3 was treated with Apt39 andthree hours later with Apt7AD. Doses were escalated through twoquantities of test articles, with the first dose occurring on Day 4 ofthe study and the second dose occurring on Day 13. To better understandthe dose-response to aptamer, the four monkeys assigned to Group 1 weresubdivided into two groups at Day 13, with two animals receiving a lowdose (Group 1a, 5 mg/kg) and two animals receiving a high dose (Group1b, 30 mg/kg).

As shown in FIG. 16, administration of Apt39 at doses ranging from 5 to30 mg/kg resulted in a profound level of anticoagulation in the monkeys.The mean APTT at each dose level exceeded 60 seconds from 0.25 to 24hours following aptamer administration, which is equivalent to <0.1%normal plasma FIX levels in the monkey. There is a dose-dependentincrease in APTT in response to Apt39 administration. However, thedose-response is not immediately evident due to the fact that, up to the6-hour time point following Apt39 administration, the aptamer plasmalevel exceeded the concentration at which the in vitro APTTdose-response curve approaches a plateau (˜40-50 μg/mL; see Table B). Attimes beyond 6 hours after administration, as the aptamer concentrationdecreases below this level, the dose-response is more apparent. APTT wasfollowed until it returned to baseline in monkeys receiving 5 and 15mg/kg doses. Mean APTT returned to baseline by 120 hours at the 5-mg/kgdose level and 192 hours at the 15-mg/kg dose level, consistent withboth the in vitro APTT dose-response curve (data not shown) and theobserved half-life of approximately 12 hours in monkeys (see Table B).The whole-blood activated clotting time (ACT) data mirrored the APTTdata (data not shown).

There is an excellent correspondence between the mean Apt39concentration 24 hours post administration in the Group 1a animals andthe mean APTT of these animals. The mean aptamer concentration of theanimals treated with 5 mg/kg at 24 hours was 15.9 μg/mL and the meanAPTT was 61.1 seconds.

TABLE B Group 1 Apt39 Plasma Levels (μg/mL) Time Post Group 1 DoseLevels (animals/dose level) Injection 5 mg/kg 15 mg/kg 30 mg/kg (hours)(n = 2)* (n = 4) (n = 2)* Pre-dose 0.2 <0.04 0.2 0.25 59.8 179.8 ± 28.9465.5 3 66.6 145.6 ± 32.5 328.9 6 42.1 101.5 ± 13.4 275.3 24 15.9  51.1± 11.2 164.6 *For Day 13 dosing, animals were split into Group 1a (5mg/kg) and 1b (30 mg/kg). For these dose levels, the average plasmalevel for the two animals per dose level is reported. The Apt39 presentin Group 1a and 1b animals at the pre-dose time point is residual Apt39from the 15-mg/kg dose at Day 4. The LLOQ of the assay is <0.04 μg/mL.In the Group 2 animals treated with the antidote only, mean APTT and ACTwere not affected by antidote administration at either dose level tested(30 and 60 mg/kg). Toxicokinetic data were collected at several timepoints over the first 24 hours after administration. As shown in TableC, low, but measurable levels of the antidote were present in plasmafrom animals receiving antidote at 0.25 hours after injection of 30mg/kg on Day 4 or 60 mg/kg on Day 13. The post-dosing level of theantidote was very low by comparison to the concentration of the aptamer(in Group 1) following IV injection.

TABLE C Group 2 Apt7AD Plasma Levels (μg/mL) Time Post Apt39 Group 2Dose Levels (4 animals/dose) Injection (hours) 30 mg/kg 60 mg/kgPre-dose <0.01 <0.01   3.25 0.4 ± 0.1  0.6 ± 0.5 6 0.02 ± 0.01* <0.02*** 24 0.01 ± 0.01** <0.01*** *1 animal at <LLOQ of 0.01 includedin calculations **3 animals at <LLOQ of 0.01 included in calculations***Average of LLOQs

The APTT data from animals treated with aptamer followed by antidote 3hours later (Group 3) are shown in FIG. 17. In agreement with the datafrom animals treated with aptamer only, administration of aptamer atthese dose levels resulted in a profound level of anticoagulation, withthe mean APTTs at 0.25 and 3 hours post administration consistent withessentially complete FIX inhibition at both dose levels. Subsequentadministration of Apt7AD rapidly and completely neutralized theanticoagulant effects of Apt39 in the monkey, with the mean APTTreturning to baseline within 15 minutes following Apt7AD administration.In the Group 3 animals treated with 30/60 mg/kg Apt39/Apt7AD, the APTTwas followed for 5 days post aptamer administration. APTT data collectedover this time frame indicate the anticoagulant effects of aptamer weredurably neutralized, with no evidence of rebound anticoagulation over120 hours, or approximately 10 half-lives of aptamer in the monkey (FIG.17).

Toxicokinetic data were collected for 24 hours following Apt39administration in the Group 3 animals (Table D). For Group 3 animals,both free aptamer and complexed aptamer plasma concentrations weremeasured. Within 15 minutes of antidote administration, the meanconcentration of free aptamer decreased 5,000-10,000 fold, to levelsbelow the Lower Limit of Quantitation (LLOQ) of the assay employed.Concomitant with the decrease in free aptamer levels, the mean plasmaconcentration of complexed aptamer increased from below the LLOQ of theassay to ˜125 to 220 μg/mL at the 15/30 and 30/60 mg/kg dose levelsrespectively, indicating the rapid decrease in free Apt39 concentrationswas due to binding of Apt7AD. The concentration of free aptamer remainedbelow the LLOQ of the assay as long as 3 hours after antidoteadministration, consistent with the APTT results. At 21 hours afterantidote administration, very low levels of Apt39 were detectable inseveral animals (mean of only 0.17 μg/mL or lower).

TABLE D Group 3 Free and Complexed Apt389 Plasma Levels (μg/mL) TimePost Group 3 Dose Levels Apt39 15/30 mg/kg Apt39 + Apt7AD 30/60 mg/kgApt39 + Apt7AD Injection Free Complexed Free Complexed (hours) Apt39Apt39 Apt39 Apt39 Pre-dose <0.04 ND 0.05 ± 0.01 ND 0.25 280.2 ± 64.3 ND467.6 ± 67   ND 3.0 214.6 ± 31.8 <0.04 488.4 ± 68.6  <0.04 3.25 <0.04125.1 ± 7.9  <0.04 218.2 ± 27.2 6 <0.04 98.7 ± 20.5 <0.04 184.8 ± 28.924  0.14 ± 0.08* 8.3 ± 4.5  <0.04 ± 0.01** 22.3 ± 12  *1 animal at <LLOQof 0.04 μg/mL included in calculations **3 animals at <LLOQ of 0.04μg/mL included in calculations Apt7AD administered at t = 3 hrsimmediately after 3 hr blood draw. (ND) Not determined.

The invention has been described with reference to various specific andpreferred embodiments and techniques. It should be understood that manyvariations and modifications may be made while remaining within thespirit and scope of the invention.

1. A first isolated nucleic acid comprising a nucleic acid sequencecomprising SEQ ID NO:17.
 2. The first nucleic acid of claim 1,comprising a three dimensional structure comprising a first and a secondstem wherein the first stem comprises five nucleotides.
 3. The firstisolated nucleic acid of claim 1, wherein the first isolated nucleicacid further comprises a suicide position that becomes single strandedupon binding of an antidote oligonucleotide, wherein the antidoteoligonucleotide is complementary to the first isolated nucleic acid andwherein the antidote oligonucleotide hybridizes to the first isolatednucleic acid.
 4. An isolated nucleic acid wherein the isolated nucleicacid comprises SEQ ID NO:8.
 5. The first isolated nucleic acid of claim1, wherein one or more nucleotides are modified, wherein themodification is a 2′-O-methyl modification or a 2′-O-fluoromodification.
 6. The first isolated nucleic acid of claim 2, wherein atleast one guanine in the second stem comprises a hydroxyl sugar (2′-OH).7. The first isolated nucleic acid of claim 2, wherein at least onecytidine in the second stem comprises a 2′-fluoro modification.
 8. Thefirst isolated nucleic acid of claim 1, wherein the first isolatednucleic acid comprises modifications as presented in the sequenceselected from the group consisting of SEQ ID NOs: 34-37, SEQ IDNO:41-49, and SEQ ID NO:50-54.
 9. The first isolated nucleic acid ofclaim 1, wherein the first isolated nucleic acid is attached to a watersoluble polymer.
 10. The first isolated nucleic acid of claim 9, whereinthe water soluble polymer is polyethylene glycol.
 11. A pharmaceuticalcomposition comprising the isolated nucleic acid of claim 1 incombination with a pharmaceutically acceptable carrier.
 12. A method forinhibiting coagulation in a host in need thereof, comprisingadministering to the host a therapeutically effective amount of anucleic acid ligand comprising a SEQ ID NO:17.
 13. The method accordingto claim 12, wherein the nucleic acid ligand comprises a modifiednucleotide modified by a 2′-O-methyl modification or a 2′-O-fluoromodification.
 14. The method according to claim 12, wherein the nucleicacid ligand is selected from the group consisting of SEQ ID NO:34-37,SEQ ID NO:41-49, and SEQ ID NO:50-54.
 15. The method according to claim12, wherein the host is suffering from or at risk of suffering from adisorder selected from the group consisting of acute myocardialinfarction (heart attack), cerebrovascular accidents (stroke), ischemia,angioplasty, CABG (coronary artery bypass grafts), cardiopulmonarybypass, thrombosis in the circuit of cardiac bypass apparatus and inpatients undergoing renal dialysis, unstable angina, pulmonary embolism,deep vein thrombosis, arterial thrombosis, and disseminatedintravascular coagulation.
 16. The method according to claim 12, whereinthe administering is intravenous, subcutaneous, transdermal orparenteral administration.
 17. A method of modulating coagulation in ahost in need thereof comprising administering to the host a firstisolated nucleic acid sequence comprising SEQ ID NO:17, wherein thefirst isolated nucleic acid sequence optionally includes one or morenucleotide modifications selected from a 2′-O-methyl modification or a2′-O-fluoro modification, and administering an effective amount of asecond isolated nucleic acid sequence comprising SEQ ID NO:8, whereinthe administering of the second nucleic acid sequence is done after theadministering the first nucleic acid sequence, and wherein the secondnucleic acid sequence reverses the effects of the first nucleic acidsequence.